Anti-Jagged1 antibodies and methods of use

ABSTRACT

The invention provides anti-Jagged1 antibodies and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.14/619,930, filed Feb. 11, 2015, which claims the benefit of priority ofU.S. Provisional Application No. 61/939,110, filed Feb. 12, 2014, eachof which is incorporated by reference herein in its entirety for anypurpose.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2016-10-19_01146-0033-01US_ST25.txt” created on Oct. 19, 2016, which is174,681 bytes in size. The information in the electronic format of thesequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to anti-Jagged antibodies and methods ofusing the same.

BACKGROUND

The Notch signaling pathway regulates a diverse array of cell functions(Kopan et al., Cell 137, 216-233 (2009)). Four Notch receptors have beenidentified in mammals, i.e., Notch 1-4, that share basic structuralelements that include an extracellular domain, a transmembrane domain,and an intracellular domain. Similarly, the canonical ligands of Notchshare certain structural similarities but a number of non-canonicalligands of Notch have also been identified (Kopan et al., Cell 137,216-233 (2009)). The five canonical ligands in mammals are Delta-like 1,Delta-like 3, Delta-like 4, Jagged1 and Jagged2. Binding of a Notchligand to the extracellular domain of a Notch receptor sets a signalingcascade in motion that begins with proteolytic cleavage at theextracellular S2 site by an alpha secretase of the ADAM (a disintegrinand metalloprotease) family. Cleavage at S2 is followed by proteolyticcleavage by a gamma secretase at the intracellular S3 site, whichresults in release of the intracellular domain and downstream eventsthat ultimately activate Notch-dependent transcription factors such asHes1 and Hey.

Aberrant Notch expression and signaling has been implicated in a numberof diseases, including cancer (Koch et al., Cell. Mol. Life Sci. 64,2746-2762 (2007)). It is clear that there continues to be a need foragents that have clinical attributes that are optimal for development astherapeutic agents. The invention described herein meets this need andprovides other benefits.

SUMMARY

The invention provides anti-Jagged1 antibodies and methods of using thesame.

The present inventors unexpectedly found that anti-Jagged1 antibody A-1(see FIG. 4 and PCT Publication No. 2014/028446) is cleaved in the heavychain following heat treatment and/or freeze-thaw conditions. The poorstability of the antibody potentially reduces its value as atherapeutic. Analysis of the cleavage site revealed no known proteasecleavage motifs. It was therefore not known whether changes to theantibody sequence could reduce the observed cleavage. Further, becausethe cleavage site is in a heavy chain HVR, even if amino acid change(s)would reduce cleavage, it was not known whether such changes could bemade without significantly reducing the antibody's affinity for Jagged1.The inventors found, surprisingly, that mutation of the amino acid atposition 101 in HVR-H3 reduced cleavage with only a slight loss ofaffinity.

In some embodiments, an isolated antibody that binds to human Jagged1 isprovided, wherein the antibody comprises an HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 55 or 59, wherein X is any amino acid otherthan S. In some embodiments, the antibody comprises at least one, atleast two, at least three, at least four, or five HVRs selected fromHVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78; HVR-H2comprising the amino acid sequence of SEQ ID NO: 28 or 36; HVR-L1comprising the amino acid sequence of SEQ ID NO: 38; HVR-L2 comprisingthe amino acid sequence of SEQ ID NO: 39; and HVR-L3 comprising theamino acid sequence of SEQ ID NO: 16 or 40. In some embodiments, theantibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQID NO: 78; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36;and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55; or (b)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; HVR-H2comprising the amino acid sequence of SEQ ID NO: 36; and HVR-H3comprising the amino acid sequence of SEQ ID NO: 59; or (c) HVR-H1comprising the amino acid sequence of SEQ ID NO: 78; HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 28; and HVR-H3 comprising theamino acid sequence of SEQ ID NO: 59. In any of the embodimentsdescribed herein, the antibody may comprise (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 38; HVR-L2 comprising the amino acidsequence of SEQ ID NO: 39; and HVR-L3 comprising the amino acid sequenceof SEQ ID NO: 40; or (b) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 38; HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or(c) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; HVR-L2comprising the amino acid sequence of SEQ ID NO: 39; and HVR-L3comprising the amino acid sequence of SEQ ID NO: 16.

In some embodiments, an isolated antibody that binds to Jagged1comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:78; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; HVR-H3comprising the amino acid sequence of SEQ ID NO: 55; HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 38; HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 39; and HVR-L3 comprising the amino acidsequence of SEQ ID NO: 40; or (b) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 78; HVR-H2 comprising the amino acid sequence ofSEQ ID NO: 36; HVR-H3 comprising the amino acid sequence of SEQ ID NO:59; HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; HVR-L2comprising the amino acid sequence of SEQ ID NO: 39; and HVR-L3comprising the amino acid sequence of SEQ ID NO: 16; or (c) HVR-H1comprising the amino acid sequence of SEQ ID NO: 78; HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 28; HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 59; HVR-L1 comprising the amino acidsequence of SEQ ID NO: 38; HVR-L2 comprising the amino acid sequence ofSEQ ID NO: 39; and HVR-L3 comprising the amino acid sequence of SEQ IDNO: 16.

In some embodiments, an isolated antibody that binds to Jagged1comprises a VH sequence having at least 95% identity to the amino acidsequence of SEQ ID NO: 54, 58, or 62. In some embodiments, an isolatedantibody that binds to Jagged1 comprises a VL sequence having at least95% identity to the amino acid sequence of SEQ ID NO: 10, 26, or 34. Insome embodiments, the antibody comprises (a) a VH sequence having atleast 95% identity to the amino acid sequence of SEQ ID NO: 54 and a VLsequence having at least 95% identity to the amino acid sequence of SEQID NO: 34; or (b) a VH sequence having at least 95% identity to theamino acid sequence of SEQ ID NO: 58 and a VL sequence having at least95% identity to the amino acid sequence of SEQ ID NO: 10; or (c) a VHsequence having at least 95% identity to the amino acid sequence of SEQID NO: 62 and a VL sequence having at least 95% identity to the aminoacid sequence of SEQ ID NO: 26.

In some embodiments, an isolated antibody that binds to human Jagged1comprises (a) a VH sequence of SEQ ID NO: 54, wherein X is any aminoacid other than S, and a VL sequence of SEQ ID NO: 34; or (b) a VHsequence of SEQ ID NO: 58, wherein X is any amino acid other than S, anda VL sequence of SEQ ID NO: 10; or (c) a VH sequence of SEQ ID NO: 62,wherein X is any amino acid other than S, and a VL sequence of SEQ IDNO: 26. In some embodiments, the antibody comprises a VH sequence of SEQID NO: 54 and a VL sequence of SEQ ID NO: 34. In some embodiments, theheavy chain comprises the amino acid sequence of SEQ ID NO: 56 and thelight chain comprises the amino acid sequence of SEQ ID NO: 53. In someembodiments, the heavy chain comprises the amino acid sequence of SEQ IDNO: 57 and the light chain comprises the amino acid sequence of SEQ IDNO: 53.

In any of the embodiments described herein, X may be any amino acidother than S or H. In any of the embodiments described herein, X may beselected from A, D, E, G, I, K, L, N, Q, R T, and V. In any of theembodiments described herein, X may be T.

In some embodiments, an isolated antibody that binds human Jagged1 isprovided, comprising (a) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 78; HVR-H2 comprising the amino acid sequence of SEQ ID NO:36; HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; HVR-L1comprising the amino acid sequence of SEQ ID NO: 38; HVR-L2 comprisingthe amino acid sequence of SEQ ID NO: 39; and HVR-L3 comprising theamino acid sequence of SEQ ID NO: 40; or (b) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 78; HVR-H2 comprising the amino acidsequence of SEQ ID NO: 36; HVR-H3 comprising the amino acid sequence ofSEQ ID NO: 64; HVR-L1 comprising the amino acid sequence of SEQ ID NO:38; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; andHVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; or (c)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 78; HVR-H2comprising the amino acid sequence of SEQ ID NO: 28; HVR-H3 comprisingthe amino acid sequence of SEQ ID NO: 64; HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 38; HVR-L2 comprising the amino acidsequence of SEQ ID NO: 39; and HVR-L3 comprising the amino acid sequenceof SEQ ID NO: 16.

In some embodiments, an isolated antibody that binds human Jagged1 isprovided, comprising (a) a VH sequence of SEQ ID NO: 33 and a VLsequence of SEQ ID NO: 34; or (b) a VH sequence of SEQ ID NO: 65 and aVL sequence of SEQ ID NO: 10; or (c) a VH sequence of SEQ ID NO: 66 anda VL sequence of SEQ ID NO: 26. In some embodiments, the antibodycomprises a VH sequence of SEQ ID NO: 33 and a VL sequence of SEQ ID NO:34.

In any of the above embodiments described herein, the antibody may be amonoclonal antibody. In certain embodiments, the antibody is a human,humanized, or chimeric antibody. In any of the above embodimentsdescribed herein, the antibody may be an antibody fragment.

Any of the above embodiments may be a full-length IgG1 antibody. In someembodiments, the antibody is an IgG1 antibody lacking effector function.In some embodiments, the antibody is an IgG1 antibody comprising anN297G or N297A mutation. In some embodiments, the antibody is an IgG1antibody comprising an N297G mutation.

In some embodiments, an isolated antibody that binds to Jagged1 isprovided, wherein the heavy chain comprises the amino acid sequence ofSEQ ID NO: 51 and the light chain comprises the amino acid sequence ofSEQ ID NO: 53. In some embodiments, the heavy chain comprises the aminoacid sequence of SEQ ID NO: 52 and the light chain comprises the aminoacid sequence of SEQ ID NO: 53. In some embodiments, the antibodycomprises (a) a heavy chain comprising the amino acid sequence of SEQ IDNO: 69 and a light chain comprising the amino acid sequence of SEQ IDNO: 75; or (b) a heavy chain comprising the amino acid sequence of SEQID NO: 70 and a light chain comprising the amino acid sequence of SEQ IDNO: 76; or (c) a heavy chain comprising the amino acid sequence of SEQID NO: 79 and a light chain comprising the amino acid sequence of SEQ IDNO: 75; or (d) a heavy chain comprising the amino acid sequence of SEQID NO: 80 and a light chain comprising the amino acid sequence of SEQ IDNO: 76.

In any of the embodiments described herein, the antibody may be anantagonist of Jagged1-mediated signaling. In some embodiments, theantibody binds human and murine Jagged1. In some embodiments, theantibody binds human, murine, rat, and cynomolgus monkey Jagged1. Insome embodiments, the antibody binds Jagged1 but does not bind Jagged2.In some embodiments, the antibody binds human Jagged1 does not bindhuman Jagged2. In some embodiments, the antibody binds human and murineJagged1 but does not bind human or murine Jagged2. In some embodiments,the antibody binds human, murine, rat, and cynomolgus monkey Jagged1 butdoes not bind human, cynomolgus monkey, or murine Jagged2. In someembodiments, the antibody binds Jagged1 but does not bind Jagged2 orDLL1. In some embodiments, the antibody binds human Jagged1 but does notbind human Jagged2 or human DLL1. In some embodiments, the antibody theantibody binds human and murine Jagged1 but does not bind human ormurine Jagged2 or human or murine DLL1. In some embodiments, theantibody the antibody binds human, murine, and cynomolgus monkey Jagged1but does not bind human, murine, or cynomolgus monkey Jagged2 or human,murine, or cynomolgus monkey DLL1. In some embodiments, the antibodybinds Jagged1 but does not bind Jagged2, DLL1, or DLL4. In someembodiments, the antibody binds human Jagged1 but does not bind humanJagged2, human DLL1, or human DLL4. In some embodiments, the antibodybinds human and murine Jagged1 but does not bind human or murineJagged2, human or murine DLL1, or human or murine DLL4. In someembodiments, the antibody binds human, murine, rat, and cynomolgusmonkey Jagged1 but does not bind human, murine, or cynomolgus monkeyJagged2, human, murine, or cynomolgus monkey DLL1, or human, murine, orcynomolgus monkey DLL4.

In some embodiments, the antibody binds human Jagged1 with an affinity(Kd) of 2 nM or stronger (i.e., less than 2 nM). In some embodiments,the antibody binds human Jagged1 with an affinity (Kd) of 1.5 nM orstronger, or 1 nM or stronger, or 0.9 nM or stronger, 0.8 nM orstronger, or 0.7 nM or stronger (i.e., less than 1.5 nM, less than 1 nM,less than 0.9 nM, less than 0.8 nM, or less than 0.7 nM). In someembodiments, the antibody binds murine Jagged1 with an affinity (Kd) of2 nM or stronger (i.e., less than 2 nM). In some embodiments, theantibody binds murine Jagged1 with an affinity (Kd) of 1.5 nM orstronger, or 1 nM or stronger, or 0.9 nM or stronger, 0.8 nM orstronger, 0.7 nM or stronger, or 0.6 nM or stronger, or 0.5 nM orstronger (i.e., less than 1.5 nM, less than 1 nM, less than 0.9 nM, lessthan 0.8 nM, less than 0.7 nM, less than 0.6 nM, or less than 0.5 nM).In some embodiments, affinity (Kd) is measured using surface plasmonresonance.

In some embodiments, the antibody binds human Jagged1 with anassociation constant (k_(on)) of at least 1.0E+04/Ms, or at least1.5E+04/Ms, or at least 2.0E+04/Ms. In some embodiments, the antibodybinds human Jagged1 with a dissociation constant (k_(off)) of less than10.0E−04/s, or less than 9.0E−04/s, or less than 8.0E−04/s, or less than7.0E−04/s. In some embodiments, the antibody binds murine Jagged1 withan association constant (k_(on)) of at least 1.0E+04/Ms, or at least1.5E+04/Ms, or at least 2.0E+04/Ms, or at least 3.0E+04/Ms, or at least4.0E+04/Ms, or at least 5.0E+04/Ms, or at least 6.0E+04/Ms, or at least7.0E+04/Ms. In some embodiments, the antibody binds murine Jagged1 witha dissociation constant (k_(off)) of less than 10.0E−04/s, or less than9.0E−04/s, or less than 8.0E−04/s, or less than 7.0E−04/s. In someembodiments, association and dissociation constants are measured usingsurface plasmon resonance.

In some embodiments, the antibody binds native, folded Jagged1, but doesnot bind denatured Jagged1. In some embodiments, the antibody bindsJagged1 in an enzyme-linked immunosorbent assay (ELISA) but does notbind Jagged1 on a Western blot. In some embodiments, the antibody bindsfolded Jagged1 in an enzyme-linked immunosorbent assay (ELISA) but doesnot bind denatured Jagged1 on a Western blot. In some embodiments, theantibody binds folded Jagged1 under physiological conditions but doesnot bind denatured Jagged1. In some embodiments, the antibody reducestumor growth in a mouse xenograft model without causing weight loss. Insome embodiments, the mouse xenograft model is a liver cancer xenograftmodel. In some embodiments, tumor growth is reduced by at least 50, atleast 60, at least 70, at least 80, or at least 90 AUC/day TGI %.

In some embodiments, use of the Jagged1 antibody reduces goblet cellmetaplasia in lungs in a mouse model of airway hyperresponsiveness. Insome embodiments, administration of the antibody reduces the number ofgoblet cells in the lungs by at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, or at least 90%. In anotheraspect, the invention provides an isolated antibody that competes withany of the above embodiments for specific binding to Jagged1. In someembodiments, an antibody is that competes for binding to human Jagged1with an antibody comprising a heavy chain variable region comprising thesequence of SEQ ID NO: 33 and a light chain variable region comprisingthe sequence of SEQ ID NO: 34, wherein the antibody is not antibody A,antibody A-1, or antibody A-2.

In another aspect, the invention provides an isolated nucleic acidencoding an isolated antibody of the above embodiments. In a furtheraspect, the invention provides a host cell comprising the isolatednucleic acid encoding the antibody. In a further aspect, the inventionprovides a method of producing an antibody comprising culturing the hostcell so that the antibody is produced.

In another aspect, the invention provides an immunoconjugate comprisingan antibody of any of the above embodiments and a cytotoxic agent.

In another aspect, the invention provides a pharmaceutical formulationcomprising an antibody of any of the above embodiments and apharmaceutically acceptable carrier.

In another aspect, an antibody of any of the above embodiments isprovided for use as a medicament. In some embodiments, an antibody ofany of the above embodiments is provided for use in treating a cancer.In some embodiments, an antibody of any of the above embodiments isprovided for use in reducing cancer cell growth.

In another aspect, a method of inhibiting Jagged1-mediated signaling isprovided. In one embodiment, a method of inhibiting Jagged1-mediatedsignaling in vitro is provided. In one embodiment, a method ofinhibiting Jagged1-mediated signaling in vivo is provided.

In another aspect, a method of treating an individual having a cancercomprising administering to the individual an effective amount of anantibody of any of the above embodiments. In one embodiment, the canceris selected from the group consisting of: breast cancer, lung cancer,brain cancer, cervical cancer, colon cancer, liver cancer, bile ductcancer, pancreatic cancer, skin cancer, B-cell malignancies, and T-cellmalignancies.

The present inventors discovered that treatment with Jagged1-antibodybiases cell fate in the airways away from a secretory cell (includinggoblet cell) fate and toward a ciliated cell fate. Jagged1 signaling isimportant for maintaining the secretory cell fate, and inhibition ofJagged1 signaling prevented goblet cell metaplasia. The presentinventors also showed that the club cell-to-ciliated cell conversion isdirect and did not involve cell division (data not shown). Thistransdifferentiation of one cell type to another occurred in the adultlung and is distinct from cell fate choices that involve progenitor celldivision, such as after damage or during development. Goblet cellmetaplasia or excess mucus is a hallmark of several airway diseases,such as asthma, cystic fibrosis, COPD and Barrett's esophagus. TheseJagged inhibition results provide the basis for therapeutic applicationsinvolving use of Jagged1 or Jagged2 inhibitors for prevention orreversal of goblet cell metaplasia and for treatment of conditionscharacterized by excess mucus, as in airway diseases (e.g., asthma,COPD, cystic fibrosis) and Barrett's esophagus.

In some embodiments, provided are methods of converting a club cell to aciliated cell, comprising administering an antagonist antibody thatbinds to human Jagged1 (including, but not limited to, any of theanti-Jagged1 antibodies described herein) to an individual. In someembodiments, provided are methods of increasing conversion of club cellsinto ciliated cells, comprising administering an antagonist antibodythat binds to human Jagged1 (including, but not limited to, any of theanti-Jagged1 antibodies described herein) to an individual. In someembodiments, the club cell is present in the adult human airway (e.g.,lung). In some embodiments, the conversion occurs in the absence of clubcell division. In some embodiments, the individual has a diseaseselected from allergy, asthma, autoimmune disease, diseases associatedwith goblet cell metaplasia (e.g., in lung) and diseases associated withexcess mucus. In some embodiments, the disease is associated with gobletcell metaplasia. In some embodiments, the disease is selected fromasthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis,and Barrett's esophagus.

In some embodiments, provided are methods of decreasing the number ofgoblet cells, comprising administering an antagonist antibody that bindsto human Jagged1 (including, but not limited to, any of the anti-Jagged1antibodies described herein) to an individual. In some embodiments,provided are methods of reducing conversion of club cells into gobletcells, comprising administering an antagonist antibody that binds tohuman Jagged1 (including, but not limited to, any of the anti-Jagged1antibodies described herein) to an individual. In some embodiments, thegoblet cell(s) is present in the adult human airway (e.g., lung). Insome embodiments, the individual has a disease selected from allergy,asthma, autoimmune disease, diseases associated with goblet cellmetaplasia (e.g., in lung) and diseases associated with excess mucus. Insome embodiments, the disease is associated with goblet cell metaplasia.In some embodiments, the disease is selected from asthma, chronicobstructive pulmonary disease (COPD), cystic fibrosis, and Barrett'sesophagus.

In some embodiments, provided are methods of reducing the formation ofgoblet cells in a subject, comprising administering an antagonistantibody that binds to human Jagged1 (including, but not limited to, anyof the anti-Jagged1 antibodies described herein) to an individual. Insome embodiments, formation of goblet cells in the adult human airway(e.g., lung) is reduced. In some embodiments, the individual has adisease selected from allergy, asthma, autoimmune disease, diseasesassociated with goblet cell metaplasia (e.g., in lung) and diseasesassociated with excess mucus. In some embodiments, the disease isassociated with goblet cell metaplasia. In some embodiments, the diseaseis selected from asthma, chronic obstructive pulmonary disease (COPD),cystic fibrosis, and Barrett's esophagus.

In some embodiments, provided are methods of decreasing mucus,comprising administering an antagonist antibody that binds to humanJagged1 (including, but not limited to, any of the anti-Jagged1antibodies described herein) to an individual. In some embodiments, themucus is airway mucus. In some embodiments, the mucus is present inadult human airway (e.g., lung). In some embodiments, the individual hasa disease selected from allergy, asthma, autoimmune disease, diseasesassociated with goblet cell metaplasia (e.g., in lung) and diseasesassociated with excess mucus. In some embodiments, the disease isassociated with goblet cell metaplasia. In some embodiments, the diseaseis selected from asthma, chronic obstructive pulmonary disease (COPD),cystic fibrosis, and Barrett's esophagus.

In some embodiments, provided are methods of increasing ciliated cellnumber, comprising administering an antagonist antibody that binds tohuman Jagged1 (including, but not limited to, any of the anti-Jagged1antibodies described herein) to an individual. In some embodiments,provided are methods of increasing formation of ciliated cells,comprising administering an antagonist antibody that binds to humanJagged1 (including, but not limited to, any of the anti-Jagged1antibodies described herein) to an individual. In some embodiments, theciliated cell is present in the adult human airway (e.g., lung). In someembodiments, the individual has a disease selected from allergy, asthma,autoimmune disease, diseases associated with goblet cell metaplasia(e.g., in lung) and diseases associated with excess mucus. In someembodiments, the disease is associated with goblet cell metaplasia. Insome embodiments, the disease is selected from asthma, chronicobstructive pulmonary disease (COPD), cystic fibrosis, and Barrett'sesophagus.

In some embodiments, an antibody that binds to human Jagged1 (including,for example, any of the anti-Jagged1 antibodies described herein) isprovided for use in treating a disease selected from allergy, asthma,autoimmune disease, diseases associated with goblet cell metaplasia(e.g., in lung) and diseases associated with excess mucus is provided,comprising administering to an individual with cancer an effectiveamount of an antibody of any of the embodiments described herein. Insome embodiments, the disease is associated with goblet cell metaplasia.In some embodiments, the disease is selected from asthma, chronicobstructive pulmonary disease (COPD), cystic fibrosis, and Barrett'sesophagus.

In some embodiments, an antibody that binds to human Jagged1 (including,for example, any of the anti-Jagged1 antibodies described herein) isprovided for use in treating a disease selected from allergy, asthma,autoimmune disease, diseases associated with goblet cell metaplasia(e.g., in lung) and diseases associated with excess mucus is provided,comprising administering to an individual with cancer an effectiveamount of an antibody of any of the embodiments described herein,wherein the antibody that binds to human Jagged1 increases conversion ofclub cells to ciliated cells in the adult human airway (e.g., lung). Insome embodiments, the disease is associated with goblet cell metaplasia.In some embodiments, the disease is selected from asthma, chronicobstructive pulmonary disease (COPD), cystic fibrosis, and Barrett'sesophagus.

In some embodiments, an antibody that binds to human Jagged1 (including,for example, any of the anti-Jagged1 antibodies described herein) isprovided for use in treating a disease selected from allergy, asthma,autoimmune disease, diseases associated with goblet cell metaplasia(e.g., in lung) and diseases associated with excess mucus is provided,comprising administering to an individual with cancer an effectiveamount of an antibody of any of the embodiments described herein,wherein the antibody that binds to human Jagged1 decreases the number ofgoblet cells in the adult human airway (e.g., lung). In someembodiments, the disease is associated with goblet cell metaplasia. Insome embodiments, the disease is selected from asthma, chronicobstructive pulmonary disease (COPD), cystic fibrosis, and Barrett'sesophagus.

In some embodiments, an antibody that binds to human Jagged1 (including,for example, any of the anti-Jagged1 antibodies described herein) isprovided for use in treating a disease selected from allergy, asthma,autoimmune disease, diseases associated with goblet cell metaplasia(e.g., in lung) and diseases associated with excess mucus is provided,comprising administering to an individual with cancer an effectiveamount of an antibody of any of the embodiments described herein,wherein the antibody that binds to human Jagged1 reduces the formationof goblet cells in the adult human airway (e.g., lung). In someembodiments, the disease is associated with goblet cell metaplasia. Insome embodiments, the disease is selected from asthma, chronicobstructive pulmonary disease (COPD), cystic fibrosis, and Barrett'sesophagus.

In some embodiments, an antibody that binds to human Jagged1 (including,for example, any of the anti-Jagged1 antibodies described herein) isprovided for use in treating a disease selected from allergy, asthma,autoimmune disease, diseases associated with goblet cell metaplasia(e.g., in lung) and diseases associated with excess mucus is provided,comprising administering to an individual with cancer an effectiveamount of an antibody of any of the embodiments described herein,wherein the antibody that binds to human Jagged1 decreases mucus in theadult human airway (e.g., lung). In some embodiments, the disease isassociated with goblet cell metaplasia. In some embodiments, the diseaseis selected from asthma, chronic obstructive pulmonary disease (COPD),cystic fibrosis, and Barrett's esophagus.

In some embodiments, an antibody that binds to human Jagged1 (including,for example, any of the anti-Jagged1 antibodies described herein) isprovided for use in treating a disease selected from allergy, asthma,autoimmune disease, diseases associated with goblet cell metaplasia(e.g., in lung) and diseases associated with excess mucus is provided,comprising administering to an individual with cancer an effectiveamount of an antibody of any of the embodiments described herein,wherein the antibody that binds to human Jagged1 increases ciliated cellnumber in the adult human airway (e.g., lung). In some embodiments, thedisease is associated with goblet cell metaplasia. In some embodiments,the disease is selected from asthma, chronic obstructive pulmonarydisease (COPD), cystic fibrosis, and Barrett's esophagus.

In some embodiments, an antibody that binds to human Jagged1 (including,for example, any of the anti-Jagged1 antibodies described herein) isprovided for use in treating a disease selected from allergy, asthma,autoimmune disease, diseases associated with goblet cell metaplasia(e.g., in lung) and diseases associated with excess mucus is provided,comprising administering to an individual with cancer an effectiveamount of an antibody of any of the embodiments described herein,wherein the antibody that binds to human Jagged1 increases formation ofciliated cells in the adult human airway (e.g., lung). In someembodiments, the disease is associated with goblet cell metaplasia. Insome embodiments, the disease is selected from asthma, chronicobstructive pulmonary disease (COPD), cystic fibrosis, and Barrett'sesophagus.

In another aspect, provided are methods of (a) converting a ciliatedcell to a club cell (e.g., wherein the ciliated cell is found in theadult human airway, e.g., lung), (b) increasing mucus (e.g., airwaymucus), (c) decreasing ciliated cell number (e.g., human airway ciliatedcell), comprising administering an agonist of Jagged signaling to anindividual.

In some embodiments, a method of treating a disease selected fromallergy, asthma, autoimmune disease, diseases associated with gobletcell metaplasia (e.g., in lung) and diseases associated with excessmucus is provided, comprising administering to an individual with canceran effective amount of antibody that binds to human Jagged1 (including,for example, any of the antibodies that bind to human Jagged1 describedherein). In some embodiments, the disease is associated with goblet cellmetaplasia (e.g., in the lung). In some embodiments, the disease isselected from asthma, chronic obstructive pulmonary disease (COPD),cystic fibrosis, and Barrett's esophagus.

In any of the embodiments described herein, wherein the Jagged1 antibodyreduces tumor growth in a mouse xenograft model without causing weightloss.

In any of the embodiments described herein, the anti-Jagged1 antibodymay be conjugated to a label. In some embodiments, the label is apositron emitter. In some embodiments, the positron emitter is ⁸⁹Zr. Insome embodiments, a method of detecting human Jagged1 in a biologicalsample is provided, comprising contacting the biological sample with anantibody described herein under conditions permissive for binding of theantibody to a naturally occurring human Jagged1, and detecting whether acomplex is formed between the antibody and a naturally occurring humanJagged1 in the biological sample. In some embodiments, the biologicalsample is selected from breast cancer, lung cancer, brain cancer,cervical cancer, colon cancer, liver cancer, bile duct cancer,pancreatic cancer, skin cancer, B-cell malignancies, and T-cellmalignancies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows exemplary amino acid sequences of human and murine Jagged1protein.

FIG. 2 shows exemplary amino acid sequences of human and murine Jagged2protein.

FIGS. 3A-D show the amino acid sequences of peptides used for phageantibody library screening and selection. All proteins were expressed asa secreted protein in BEVS cells and their sequences are listed in theN-terminal to C-terminal direction. (A) Amino acid sequence of expressedprotein murine Jagged 1-DSL-EGF1-4 (Q34-D377). The bold font at theN-terminus represents a short linker sequence (ADLGS) (SEQ ID NO: 82).The bold font at the C-terminus represents a short linker sequence(EFG), a thrombin cleavage site (LVPRGS) (SEQ ID NO: 83), a G spacer andthe 6-His tag. (B) Amino acid sequence of expressed protein humanJag1-DSL-EGF1-4. Only the Jag1 sequence is shown although the antigenalso contained a TEV protease cleavage site and 6-His tag at theC-terminus. (C) Amino acid sequence of expressed protein murineJag2-DSL-EGF1-4 (M27-E388). The bold font at the N-terminus represents ashort linker sequence (ADLGS) (SEQ ID NO: 82). The bold font at theC-terminus represents a short linker sequence (EFG), a thrombin cleavagesite (LVPRGS) (SEQ ID NO: 83), a G spacer and the 6-His tag. (D) Aminoacid sequence of expressed protein human Jag2-DSL-EGF1-4 (R2-E388). Thebold font at the C-terminus represents a short linker sequence (EFG), athrombin cleavage site (LVPRGS) (SEQ ID NO: 83), a G spacer and the6-His tag.

FIGS. 4A-1-B-2 show an alignment of the amino acid sequences for theheavy (FIG. 4A-1 and FIG. 4A-2) and light (FIG. 4B-1 and FIG. 4B-2)chain variable domains of anti-Jagged1 (A, A-1, A-2), anti-Jagged2 (B,B-1, B-2, B-3), and anti-Jagged1/2 antibodies (C, C-1, D, D-1, D-2, D-3,D-4, D-5). Amino acid positions of the complementarity determiningregions (CDRs) are indicated.

FIGS. 5A-B show the H1, H2, and H3 heavy chain hypervariable region(HVR) sequences of anti-Jagged1 antibodies, as described in theExamples. Amino acid positions are numbered according to the Kabatnumbering system as described herein and elsewhere.

FIG. 6 shows the L1, L2, and L3 light chain HVR sequences ofanti-Jagged1 antibodies described in the Examples. Amino acid positionsare numbered according to the Kabat numbering system as described below.

FIG. 7 shows light and heavy chain framework sequences of anti-Jagged1antibodies described in the Examples. Numbers in superscript indicateamino acid positions according to Kabat.

FIG. 8 shows binding specificity of antibodies obtained from thescreening. Results of ELISA assays measuring binding specificity ofAntibodies A and B, identified during screening using humanJag1-DSL-EGF1-4 (FIG. 3B) for antibody A and murine and humanJag2-DSL-EGF1-4 (FIGS. 3C and D) for antibody B. Black columns=bindingto human Jagged1; gray columns=binding to human Jagged2. C-1 is anantibody that binds to both Jagged1 and Jagged2.

FIGS. 9A-B show inhibition of Notch signaling by affinity maturedanti-Jagged antibodies. Co-culture assays were performed as described inExample 3. Phage antibodies at the indicated concentration are indicatedon the x-axis. DAPT at the indicated concentrations served as positivecontrol for inhibition of Notch signaling; DMSO served as vehiclecontrol. Signaling was induced by Jagged1 (dark gray columns) or byJagged2 (light gray columns). Untreated=cultures that were notstimulated with ligand and not treated with antibody; NoStimulation=cultures not stimulated with ligand; agD=isotype controlantibody; Stim/no AB=cultures stimulated with ligand but not treatedwith antibody; gamma-secretase inhibitor DAPT or the DAPT vehiclecontrol of DMSO.

FIGS. 10A-B show that combined inhibition of Jagged1 and Jagged2 causesrapid weight loss. (A) Mice were dosed twice per week with theanti-Jagged1/2 antibody C-1 (anti-J1/2; 5-10 mpk), the anti-Jagged1antibody A-2 (anti-11; 5-20 mpk), the anti-Jagged2 antibody B-3(anti-J2; 5-20 mpk), the antibody A-2 and B-3 together (anti-J1 & -2; 5mpk each) or an isotype control antibody (20 mpk). Total antibodyconcentration of each dosing was brought up to 20 mpk with the isotypecontrol antibody, where necessary. The average body weight changes(y-axis) are graphed as a percentage of starting body weight over time(x-axis). (B) Balb/c mice (ten per group, individually housed) wereinjected IP twice per week with either 30 mpk of anti-gD isotype controlantibody or with a combination of 15 mpk antibody A-2 plus 15 mpkantibody B-3 for eight days. Food intake was assessed by daily weighingof the food delivered and remaining in each cage. Error bars representstandard deviations (n=10).

FIGS. 11A-1-B-2 show inhibition of human lung cancer cell growth by ananti-Jagged1 antagonist antibody in vivo. Mice bearing human lung cancerxenografts were injected twice per week intraperitoneally (IP) with 20mpk anti-gD isotype control antibody (Isotype control Ab) or withanti-Jagged1 antibody A-2 (Anti-Jag1), with the injections startingafter average tumor volumes (measured with calipers) reachedapproximately 180 mm³. Tumor volumes (y-axis) were subsequently measuredfor 19 days. FIG. 11A-1 and FIG. 11A-2: The average tumor volumes foreach group (n=10) were plotted over time (x-axis) using a linear mixedeffects model (FIG. 11A-1). Tumor volumes for each mouse in each groupare depicted in the two panels in FIG. 11A-2. FIG. 11B-1 and FIG. 11B-2:Total body weight of each mouse was measured and graphed as thepercentage change averaged for each group (FIG. 11B-1) or for each mousein each group (FIG. 11B-2).

FIGS. 12A-B show inhibition of human breast cancer cell growth byanti-Jagged1 and anti-Jagged2 antagonist antibodies in vivo. C.B-17SCID.bg mice with human breast cancer xenografts were injected on days0, 4, 7, 12, 15, 18, 22, 25, 29, 32, 36, 43, 50, and 57 with anti-gDisotype control antibody (Anti-gD), anti-ragweed isotype controlantibody (anti-ragweed), anti-Jagged1 antibody A-2 in the human IgG1backbone (anti-Jag1 A-2 (hIgG1)), anti-Jagged1 antibody A-2 in themurine IgG2a backbone (anti-Jag1 A-2 (mIgG2a)), or anti-Jagged2 antibodyB-3 in the human IgG1 backbone (anti-Jag2 B-3 (hIgG1)). Tumor volumes(y-axis) of treatment groups (A) or individual animals (B) were plottedusing a linear mixed effects model over time (x-axis).

FIGS. 13A-C show cleavage of the anti-Jagged1 antibodies in the heavychain. (A) Antibodies A, A-1 and A-2 were analyzed by standard SDS-PAGEand protein staining methods in the absence (−) or presence (+) of thereducing agent DTT, as indicated. Molecular mass standards (in kD) arealso shown. Panel (B) displays a representative scan of a massspectrometry (MS) analysis (LC-MS/TOF, reducing conditions) of A-1. Thepositions of the relevant fragments (peaks) are annotated, and themolecular masses are shown above the peaks. This analysis indicated thatthe cleavage site was between HC amino acids G100 and S101 in CDR3, asdiagramed in the HC amino acid sequence in panel (C), with the arrowmarking the cleavage position.

FIGS. 14A-C shows N-terminal peptide sequencing of the anti-Jagged1antibody heavy chain cleavage site. Following the standard methods forSDS-PAGE under reducing conditions and protein staining, the full-lengthand cleaved fragments of the indicated antibody preparations wereseparated and identified (A). The C-terminal (B) and N-terminal (C)cleaved peptide fragments were cut out of the gel and sequenced usingstandard methods for peptide N-terminal sequencing to precisely identifythe site of cleavage. The underlined sequences in panels (B) and (C)note the N-terminal sequences of each of the peptides sequenced, withthe sequence in panel (B) marking the cleavage site and the sequence inpanel (C) marking the N-terminus of the HC.

FIG. 15 shows the effect of amino acid changes at heavy chain S101 ofthe anti-Jagged1 antibodies on heavy chain cleavage.

FIGS. 16A-B show (A) SDS-PAGE analysis of cleavage of anti-Jagged1antibodies incubated at 70° C.; and (B) a summary of the percent heavychain cleavage observed for each antibody preparation at 70° C. and at95° C.

FIGS. 17A-B show (A) SDS-PAGE analysis of cleavage of anti-Jagged1antibody A-1 after varying numbers of freeze-thaw cycles, and (B) thepercent heavy chain cleavage observed under each condition.

FIGS. 18A-C show (A) inhibition of Jagged1-induced activation byanti-Jagged1 antibodies A-1 (left bars) and A-1(S101T) (right bars) atvarious concentrations. Panels (B) and (C) shows average fireflyluciferase values and average Renilla luciferase values, respectively,which were used to calculate the data in (A), as described in Example10.

FIG. 19 shows immunofluorescent staining of ciliated cells (as marked byimmunofluorescent detection of alpha-tubulin in red) and club cells (asmarked by immunofluorescent detection of CC10 in green) in thebronchiolar epithelium of mice administered anti-Jagged2 antibodies incombination with anti-Jagged1 antibody A-1 or A-1(S101T), or an isotypecontrol, as described in Example 11.

FIGS. 20A-B show (A) a LME (linear mixed effects) graph of tumor volumein a liver cancer xenograft model mice treated with anti-Jagged1antibody A-1 or A-1(S101T), and (B) the treatment groups shown in (A)and the dose, tumor volume on the last day of the study (day 44),AUC/Day % TGI (area under the curve per day percentage tumor growthinhibition (TGI) relative to control, in which lower and upper refer tothe lowest and uppermost, respectively, % TGI values for individualanimals in each group), tumor doubling time in days (TTP 2×), and thenumber of mice showing a partial response during the experiment (PR).

FIGS. 21A-B show (A) a LME (linear mixed effects) graph of mouse bodyweight over time for the mice shown in FIG. 20, and (B) the treatmentgroups shown in (A) and the dose, % change in body weight on the lastday of the study (% BW Last Day), the maximum % change in body weight(Max % BW), and the day on which the maximum change in bodyweightoccurred (Max % BW Day), and the (AUC/Day (lower,upper)).

FIGS. 22A-B show the amino acid sequences for the (A) heavy chain and(B) light chain variable domains of anti-Jagged1 antibody A-1(S101T).Amino acid positions of the complementarity determining regions (CDRs)are indicated.

FIGS. 23A-C show (A) Periodic Acid-Schiff staining of lung airways ofcontrol, anti-Jagged1, anti-Jagged2 or the combination ofanti-Jagged1+anti-Jagged2 treated mice, (B) quantification of the numberof goblet cells in the airways of the different treatment groups, and(C) inflammation index as assessed be H&E staining.

FIG. 24 shows binding of (left) anti-Jagged1 antibody A-2 and (right)anti-Jagged1/2 antibody C-1, to human Jagged1, murine Jagged1, humanJagged 2, murine Jagged2, human DLL1, murine DLL1, human DLL4 and murineDLL4.

FIG. 25 shows clearance of anti-Jagged1 A-1-S101T antibody following asingle intravenous administration of three different doses of theantibody in mice.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-Jagged antibody” and “an antibody that binds to Jagged”refer to an antibody that is capable of binding Jagged1, Jagged2, orJagged1 and 2 (Jagged1/2) with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetingJagged. In one embodiment, the extent of binding of an anti-Jaggedantibody to an unrelated, non-Jagged protein is less than about 10% ofthe binding of the antibody to Jagged as measured, e.g., by aradioimmunoassay (MA). In certain embodiments, an antibody that binds toJagged has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸Mto 10⁻¹³M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certain embodiments, ananti-Jagged antibody binds to an epitope of Jagged that is conservedamong Jagged from different species. The terms “anti-Jagged1 antibody”and “an antibody that binds to Jagged1” refer to an antibody that iscapable of binding Jagged1 with sufficient affinity such that theantibody is useful as a diagnostic and/or therapeutic agent in targetingJagged1.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

As used herein, “asthma” refers to a complex disorder characterized byvariable and recurring symptoms, reversible airflow obstruction (e.g.,by bronchodilator) and bronchial hyperresponsiveness which may or maynot be associated with underlying inflammation. Examples of asthmainclude aspirin sensitive/exacerbated asthma, atopic asthma, severeasthma, mild asthma, moderate to severe asthma, corticosteroid naïveasthma, chronic asthma, corticosteroid resistant asthma, corticosteroidrefractory asthma, newly diagnosed and untreated asthma, asthma due tosmoking, asthma uncontrolled on corticosteroids and other asthmas asmentioned in J Allergy Clin Immunol (2010) 126(5):926-938.

A “blocking” antibody or an “antagonist” antibody is one whichsignificantly inhibits (either partially or completely) a biologicalactivity of the antigen it binds.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Generally, antibodiescomprise six HVRs: three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97(L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991));

(c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55(L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum etal. J. Mol. Biol. 262: 732-745 (1996)); and

(d) combinations of (a), (b), and/or (c), including HVR amino acidresidues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1),26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

Unless otherwise indicated, HVR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-Jagged antibody” refers to oneor more nucleic acid molecules encoding antibody heavy and light chains(or fragments thereof), including such nucleic acid molecule(s) in asingle vector or separate vectors, and such nucleic acid molecule(s)present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “Jagged” or “Jag,” as used herein, refers to any native Jaggedfrom any vertebrate source, including mammals such as primates (e.g.humans) and rodents (e.g., mice and rats), unless otherwise indicated.The term encompasses “full-length,” unprocessed Jagged as well as anyform of Jagged that results from processing in the cell. The term alsoencompasses naturally occurring variants of Jagged, e.g., splicevariants or allelic variants. The amino acid sequence of an exemplaryhuman and murine Jagged1 and Jagged2 is shown in FIGS. 1 and 2 (SEQ IDNOS:1-4), respectively.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

II. Compositions and Methods

In one aspect, the invention is based, in part, on the identification ofanti-Jagged antibodies and fragments thereof. In certain embodiments,antibodies that bind to at least one Jagged are provided. Antibodies ofthe invention are useful, e.g., for the diagnosis or treatment ofcancer. Accordingly, the invention provides methods, compositions, kits,and articles of manufacture related to anti-Jagged antibodies.

A. Exemplary Anti-Jagged1 Antibodies

In one aspect, the invention provides isolated antibodies that bind toJagged1.

In some embodiments, the antibody is an antagonist of Jagged1-mediatedsignaling. In some embodiments, the antibody binds human and murineJagged1. In some embodiments, the antibody binds human, murine, andcynomolgus monkey Jagged1. In some embodiments, the antibody bindsJagged1 but does not bind Jagged2. In some embodiments, the antibodybinds human Jagged1 does not bind human Jagged2. In some embodiments,the antibody binds human and murine Jagged1 but does not bind human ormurine Jagged2. In some embodiments, the antibody binds human, murine,and cynomolgus monkey Jagged1 but does not bind human, cynomolgusmonkey, or murine Jagged2. In some embodiments, the antibody bindsJagged1 but does not bind Jagged2 or DLL1. In some embodiments, theantibody binds human Jagged1 but does not bind human Jagged2 or humanDLL1. In some embodiments, the antibody the antibody binds human andmurine Jagged1 but does not bind human or murine Jagged2 or human ormurine DLL1. In some embodiments, the antibody the antibody binds human,murine, and cynomolgus monkey Jagged1 but does not bind human, murine,or cynomolgus monkey Jagged2 or human, murine, or cynomolgus monkeyDLL1. In some embodiments, the antibody binds Jagged1 but does not bindJagged2, DLL1, or DLL4. In some embodiments, the antibody binds humanJagged1 but does not bind human Jagged2, human DLL1, or human DLL4. Insome embodiments, the antibody binds human and murine Jagged1 but doesnot bind human or murine Jagged2, human or murine DLL1, or human ormurine DLL4. In some embodiments, the antibody binds human, murine, andcynomolgus monkey Jagged1 but does not bind human, murine, or cynomolgusmonkey Jagged2, human, murine, or cynomolgus monkey DLL1, or human,murine, or cynomolgus monkey DLL4.

In some embodiments, the antibody binds human Jagged1 with an affinity(Kd) of 2 nM or stronger (i.e., less than 2 nM). In some embodiments,the antibody binds human Jagged1 with an affinity (Kd) of 1.5 nM orstronger, or 1 nM or stronger, or 0.9 nM or stronger, 0.8 nM orstronger, or 0.7 nM or stronger (i.e., less than 1.5 nM, less than 1 nM,less than 0.9 nM, less than 0.8 nM, or less than 0.7 nM). In someembodiments, the antibody binds murine Jagged1 with an affinity (Kd) of2 nM or stronger (i.e., less than 2 nM). In some embodiments, theantibody binds murine Jagged1 with an affinity (Kd) of 1.5 nM orstronger, or 1 nM or stronger, or 0.9 nM or stronger, 0.8 nM orstronger, 0.7 nM or stronger, or 0.6 nM or stronger, or 0.5 nM orstronger (i.e., less than 1.5 nM, less than 1 nM, less than 0.9 nM, lessthan 0.8 nM, less than 0.7 nM, less than 0.6 nM, or less than 0.5 nM).

In some embodiments, the antibody binds native, folded Jagged1, but doesnot bind denatured Jagged1. In some embodiments, the antibody bindsJagged1 in an enzyme-linked immunosorbent assay (ELISA) but does notbind Jagged1 on a Western blot. In some embodiments, the antibody bindsfolded Jagged1 in an enzyme-linked immunosorbent assay (ELISA) but doesnot bind denatured Jagged1 on a Western blot. In some embodiments, theantibody binds folded Jagged1 under physiological conditions but doesnot bind denatured Jagged1. “Native, folded” Jagged1 refers to Jagged1that has undergone protein folding under physiological conditions andhas been maintained in a folded state. In some embodiments, Jagged1 hasbeen maintained in a folded state in solution.

In some embodiments, the invention provides an anti-Jagged1 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or78; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28 or36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55 or59, wherein X is any amino acid other than S; (d) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 16 or 40. In some embodiments, the inventionprovides an anti-Jagged1 antibody comprising at least one, two, three,four, five, or six HVRs selected from (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 35 or 78; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 36; (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 55, wherein X is any amino acid other than S; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 40. In someembodiments, the invention provides an anti-Jagged1 antibody comprisingat least one, two, three, four, five, or six HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 59, wherein X is anyamino acid other than S; (d) HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 38; (e) HVR-L2 comprising the amino acid sequence of SEQID NO: 39; and (f) HVR-L3 comprising the amino acid sequence of SEQ IDNO: 16. In some embodiments, the invention provides an anti-Jagged1antibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 35 or 78; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 36; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59,wherein X is any amino acid other than S; (d) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 38; (e) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 39; and (f) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 16. In some embodiments, X is any amino acidother than S. In some embodiments, X is any amino acid other than S orH. In some embodiments, X is selected from A, D, E, G, I, K, L, N, Q, RT, and V. In some embodiments, X is T.

In some embodiments, the invention provides an anti-Jagged1 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or78; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 37; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 40. In someembodiments, the invention provides an anti-Jagged1 antibody comprisingat least one, two, three, four, five, or six HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 64; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16. In someembodiments, the invention provides an anti-Jagged1 antibody comprisingat least one, two, three, four, five, or six HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 64; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 38; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28 or 36; and(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55 or 59,wherein X is any amino acid other than S. In one embodiment, theantibody comprises HVR-H3 comprising the amino acid sequence of SEQ IDNO: 55 or 59, wherein X is any amino acid other than S. In anotherembodiment, the antibody comprises HVR-H3 comprising the amino acidsequence of SEQ ID NO: 55 or 59, wherein X is any amino acid other thanS and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16 or 40.In a further embodiment, the antibody comprises HVR-H3 comprising theamino acid sequence of SEQ ID NO: 55 or 59, wherein X is any amino acidother than S, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16or 40, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28 or36. In a further embodiment, the antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 35 or 78; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 28 or 36; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55 or 59,wherein X is any amino acid other than S. In some embodiments, X is anyamino acid other than S. In some embodiments, X is any amino acid otherthan S or H. In some embodiments, X is selected from A, D, E, G, I, K,L, N, Q, R T, and V. In some embodiments, X is T.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55, wherein X isany amino acid other than S. In one embodiment, the antibody comprisesHVR-H3 comprising the amino acid sequence of SEQ ID NO: 55, wherein X isany amino acid other than S. In another embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55,wherein X is any amino acid other than S and HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 40. In a further embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55,wherein X is any amino acid other than S, HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 40, and HVR-H2 comprising the amino acidsequence of SEQ ID NO: 36. In a further embodiment, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35or 78; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36;and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 55,wherein X is any amino acid other than S. In some embodiments, X is anyamino acid other than S. In some embodiments, X is any amino acid otherthan S or H. In some embodiments, X is selected from A, D, E, G, I, K,L, N, Q, R T, and V. In some embodiments, X is T. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 35 or 78; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 36; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 37.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, wherein X isany amino acid other than S. In one embodiment, the antibody comprisesHVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, wherein X isany amino acid other than S. In another embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59,wherein X is any amino acid other than S and HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 16. In a further embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59,wherein X is any amino acid other than S, HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 16, and HVR-H2 comprising the amino acidsequence of SEQ ID NO: 28. In a further embodiment, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35or 78; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28;and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59,wherein X is any amino acid other than S. In some embodiments, X is anyamino acid other than S. In some embodiments, X is any amino acid otherthan S or H. In some embodiments, X is selected from A, D, E, G, I, K,L, N, Q, R T, and V. In some embodiments, X is T. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 35 or 78; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 28; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 64.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, wherein X isany amino acid other than S. In one embodiment, the antibody comprisesHVR-H3 comprising the amino acid sequence of SEQ ID NO: 59, wherein X isany amino acid other than S. In another embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59,wherein X is any amino acid other than S and HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 16. In a further embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59,wherein X is any amino acid other than S, HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 16, and HVR-H2 comprising the amino acidsequence of SEQ ID NO: 36. In a further embodiment, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35or 78; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36;and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 59,wherein X is any amino acid other than S. In some embodiments, X is anyamino acid other than S. In some embodiments, X is any amino acid otherthan S or H. In some embodiments, X is selected from A, D, E, G, I, K,L, N, Q, R T, and V. In some embodiments, X is T. In a furtherembodiment, the antibody comprises (a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 35 or 78; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 36; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 64.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16 or 40. In oneembodiment, the antibody comprises (a) HVR-L1 comprising the amino acidsequence of SEQ ID NO: 38; (b) HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 39; and (c) HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 16 or 40. In one embodiment, the antibody comprises (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment,the antibody comprises (a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 38; (b) HVR-L2 comprising the amino acid sequence of SEQ IDNO: 39; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:40.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 35 or 78, (ii) HVR-H2 comprising the amino acid sequence ofSEQ ID NO: 28 or 36, and (iii) HVR-H3 comprising an amino acid sequenceselected from SEQ ID NO: 55 or 59, wherein X is any amino acid otherthan S; and (b) a VL domain comprising at least one, at least two, orall three VL HVR sequences selected from (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 38, (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 39, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 16 or 40. In another aspect, an antibody of theinvention comprises (a) a VH domain comprising at least one, at leasttwo, or all three VH HVR sequences selected from (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 35 or 78, (ii) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 36, and (iii) HVR-H3 comprising anamino acid sequence selected from SEQ ID NO: 55, wherein X is any aminoacid other than S; and (b) a VL domain comprising at least one, at leasttwo, or all three VL HVR sequences selected from (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 38, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 39, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 40. In another aspect, an antibody ofthe invention comprises (a) a VH domain comprising at least one, atleast two, or all three VH HVR sequences selected from (i) HVR-H1comprising the amino acid sequence of SEQ ID NO: 35 or 78, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO: 28, and (iii) HVR-H3comprising an amino acid sequence selected from SEQ ID NO: 59, wherein Xis any amino acid other than S; and (b) a VL domain comprising at leastone, at least two, or all three VL HVR sequences selected from (i)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39, and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 16. In another aspect,an antibody of the invention comprises (a) a VH domain comprising atleast one, at least two, or all three VH HVR sequences selected from (i)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78, (ii)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, and (iii)HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 59,wherein X is any amino acid other than S; and (b) a VL domain comprisingat least one, at least two, or all three VL HVR sequences selected from(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (ii)HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16. In someembodiments, X is any amino acid other than S. In some embodiments, X isany amino acid other than S or H. In some embodiments, X is selectedfrom A, D, E, G, I, K, L, N, Q, R T, and V.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising (i) HVR-H1 comprising the amino acid sequence of SEQID NO: 35 or 78, (ii) HVR-H2 comprising the amino acid sequence of SEQID NO: 28 or 36, and (iii) HVR-H3 comprising an amino acid sequenceselected from SEQ ID NO: 55 or 59, wherein X is any amino acid otherthan S; and (b) a VL domain comprising (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 38, (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 39, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 16 or 40. In another aspect, an antibody of theinvention comprises (a) a VH domain comprising (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO: 35 or 78, (ii) HVR-H2 comprising theamino acid sequence of SEQ ID NO: 36, and (iii) HVR-H3 comprising anamino acid sequence selected from SEQ ID NO: 55, wherein X is any aminoacid other than S; and (b) a VL domain comprising (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 38, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 39, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 40. In another aspect, an antibody ofthe invention comprises (a) a VH domain comprising (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 35 or 78, (ii) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 28, and (iii) HVR-H3 comprising anamino acid sequence selected from SEQ ID NO: 59, wherein X is any aminoacid other than S; and (b) a VL domain comprising (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 38, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 39, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 16. In another aspect, an antibody ofthe invention comprises (a) a VH domain comprising (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 35 or 78, (ii) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 36, and (iii) HVR-H3 comprising anamino acid sequence selected from SEQ ID NO: 59, wherein X is any aminoacid other than S; and (b) a VL domain comprising (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 38, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 39, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 16. In some embodiments, X is anyamino acid other than S. In some embodiments, X is any amino acid otherthan S or H. In some embodiments, X is selected from A, D, E, G, I, K,L, N, Q, R T, and V.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 35 or 78, (ii) HVR-H2 comprising the amino acid sequence ofSEQ ID NO: 36, and (iii) HVR-H3 comprising an amino acid sequenceselected from SEQ ID NO: 37; and (b) a VL domain comprising at leastone, at least two, or all three VL HVR sequences selected from (i)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39, and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 40. In another aspect,an antibody of the invention comprises (a) a VH domain comprising atleast one, at least two, or all three VH HVR sequences selected from (i)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78, (ii)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and (iii)HVR-H3 comprising an amino acid sequence selected from SEQ ID NO: 64;and (b) a VL domain comprising at least one, at least two, or all threeVL HVR sequences selected from (i) HVR-L1 comprising the amino acidsequence of SEQ ID NO: 38, (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 39, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 16. In another aspect, an antibody of theinvention comprises (a) a VH domain comprising at least one, at leasttwo, or all three VH HVR sequences selected from (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 35 or 78, (ii) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 36, and (iii) HVR-H3 comprising anamino acid sequence selected from SEQ ID NO: 64; and (b) a VL domaincomprising at least one, at least two, or all three VL HVR sequencesselected from (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 38, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39,and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising (i) HVR-H1 comprising the amino acid sequence of SEQID NO: 35 or 78, (ii) HVR-H2 comprising the amino acid sequence of SEQID NO: 36, and (iii) HVR-H3 comprising an amino acid sequence selectedfrom SEQ ID NO: 37; and (b) a VL domain comprising (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 38, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO: 39, and (c) HVR-L3 comprising theamino acid sequence of SEQ ID NO: 40. In another aspect, an antibody ofthe invention comprises (a) a VH domain comprising (i) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 35 or 78, (ii) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 28, and (iii) HVR-H3 comprising anamino acid sequence selected from SEQ ID NO: 64; and (b) a VL domaincomprising (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:38, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39, and(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16. Inanother aspect, an antibody of the invention comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:35 or 78, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:36, and (iii) HVR-H3 comprising an amino acid sequence selected from SEQID NO: 64; and (b) a VL domain comprising (i) HVR-L1 comprising theamino acid sequence of SEQ ID NO: 38, (ii) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 39, and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 16.

In some embodiments, an anti-Jagged1 antibody comprises (a) a VH domaincomprising (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:35 or 78, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:71, wherein X1 is selected from P and G, X2 is selected from D and N,and X3 is selected from T and S, and (iii) HVR-H3 comprising an aminoacid sequence selected from SEQ ID NO: 72, wherein X1 is any amino acidother than S, and X2 is W or L; and (b) a VL domain comprising (i)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO: 39, and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 74, wherein X1 is S orY, X2 is P or A, and X3 is P or T. In some embodiments, the X1 in SEQ IDNO: 72 is any amino acid other than S or H. In some embodiments, the X1in SEQ ID NO: 72 is selected from A, D, E, G, I, K, L, N, Q, R T, and V.In some embodiments, the X1 in SEQ ID NO: 72 is T.

In one embodiment, an anti-Jagged1 antibody comprises HVRs as in any ofthe above embodiments, and further comprises an acceptor humanframework, e.g. a human immunoglobulin framework or a human consensusframework. In another embodiment, an anti-Jagged1 antibody comprisesHVRs as in any of the above embodiments, and further comprises a VHcomprising at least one, two, three, or four FRs selected from an FR1comprising the amino acid sequence of SEQ ID NO: 47; an FR2 comprisingthe amino acid sequence of SEQ ID NO: 48; an FR3 comprising the aminoacid sequence of SEQ ID NO: 49; and an FR4 comprising the amino acidsequence of SEQ ID NO: 50. In another embodiment, an anti-Jagged1antibody comprises HVRs as in any of the above embodiments, and furthercomprises a VL comprising at least one, two, three, or four FRs selectedfrom an FR1 comprising the amino acid sequence of SEQ ID NO: 43; an FR2comprising the amino acid sequence of SEQ ID NO: 44; an FR3 comprisingthe amino acid sequence of SEQ ID NO: 45; and an FR4 comprising theamino acid sequence of SEQ ID NO: 46.

In another aspect, an anti-Jagged1 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence of SEQ ID NO: 54, 58, or 62, wherein X is any amino acid otherthan S. In some such embodiments, the VH sequence comprises an HVR-H3 ofSEQ ID NO: 55 or 59, wherein X is any amino acid other than S. In someembodiments, the VH comprises one, two or three HVRs selected from: (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 35 or 78, (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 55 or 59,wherein X is any amino acid other than S, and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 37 or 64. In some embodiments, X isany amino acid other than S. In some embodiments, X is any amino acidother than S or H. In some embodiments, X is selected from A, D, E, G,I, K, L, N, Q, R T, and V. In some embodiments, X is T. In anotheraspect, an anti-Jagged1 antibody comprises a heavy chain variable domain(VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO: 33, 65, or 66. In some such embodiments, the VH sequence comprisesan HVR-H3 of SEQ ID NO: 37 or 64. In some embodiments, the VH comprisesone, two or three HVRs selected from: (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 35 or 78, (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO: 28 or 36, and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 37 or 64. In certain embodiments, a VHsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-Jagged1 antibody comprising that sequence retains the ability tobind to at least one Jagged1. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). In certain embodiments, an anti-Jagged1 antibody comprises a heavychain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 33. In some such embodiments, the VHsequence comprises an HVR-H3 of SEQ ID NO: 37. In some embodiments, theVH comprises one, two or three HVRs selected from: (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO: 35 or 78, (b) HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 36, and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 37. In some embodiments, theanti-Jagged1 antibody comprises the VH sequence in SEQ ID NO: 33, 65, or66, including post-translational modifications of that sequence. In someembodiments, the anti-Jagged1 antibody comprises the VH sequence in SEQID NO: 33, including post-translational modifications of that sequence.In some embodiments, the anti-Jagged1 antibody comprises the VH sequencein SEQ ID NO: 65, including post-translational modifications of thatsequence. In some embodiments, the anti-Jagged1 antibody comprises theVH sequence in SEQ ID NO: 66, including post-translational modificationsof that sequence.

In another aspect, an anti-Jagged1 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 10, 26, or 34. Incertain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-Jagged1 antibody comprising thatsequence retains the ability to bind to Jagged1. In certain embodiments,a total of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO: 10, 26, or 34. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). In some embodiments, the VL comprises one, twoor three HVRs selected from (a) HVR-L1 comprising the amino acidsequence of SEQ ID NO: 38; (b) HVR-L2 comprising the amino acid sequenceof SEQ ID NO: 39; and (c) HVR-L3 comprising the amino acid sequence ofSEQ ID NO: 16 or 40. In some embodiments, the anti-Jagged1 antibodycomprises the VL sequence in SEQ ID NO: 10, 26, or 34, includingpost-translational modifications of that sequence. In some embodiments,the anti-Jagged1 antibody comprises the VL sequence in SEQ ID NO: 34,including post-translational modifications of that sequence. In someembodiments, the anti-Jagged1 antibody comprises the VL sequence in SEQID NO: 10, including post-translational modifications of that sequence.In some embodiments, the anti-Jagged1 antibody comprises the VL sequencein SEQ ID NO: 26, including post-translational modifications of thatsequence.

In another aspect, an anti-Jagged1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO: 54, wherein Xis any amino acid other than S; and SEQ ID NO: 34, respectively,including post-translational modifications of those sequences. In oneembodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:58, wherein X is any amino acid other than S; and SEQ ID NO: 10,respectively, including post-translational modifications of thosesequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 62, wherein X is any amino acid other than S;and SEQ ID NO: 26, respectively, including post-translationalmodifications of those sequences. In some embodiments, X is any aminoacid other than S. In some embodiments, X is any amino acid other than Sor H. In some embodiments, X is selected from A, D, E, G, I, K, L, N, Q,R T, and V. In some embodiments, X is T.

In another aspect, an anti-Jagged1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO: 33 and SEQ IDNO: 34, respectively, including post-translational modifications ofthose sequences. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO: 65 and SEQ ID NO: 10, respectively, includingpost-translational modifications of those sequences. In one embodiment,the antibody comprises the VH and VL sequences in SEQ ID NO: 66 and SEQID NO: 26, respectively, including post-translational modifications ofthose sequences.

In some embodiments, an anti-Jagged1 antibody comprises a heavy chaincomprising the sequence of SEQ ID NO: 57, wherein X is any amino acidother than S, and a light chain comprising the sequence of SEQ ID NO:53. In some embodiments, an anti-Jagged1 antibody comprises a heavychain comprising the sequence of SEQ ID NO: 67, wherein X is any aminoacid other than S, and a light chain comprising the sequence of SEQ IDNO: 75. In some embodiments, an anti-Jagged1 antibody comprises a heavychain comprising the sequence of SEQ ID NO: 68, wherein X is any aminoacid other than S, and a light chain comprising the sequence of SEQ IDNO: 76. In some embodiments, X is any amino acid other than S. In someembodiments, X is any amino acid other than S or H. In some embodiments,X is selected from A, D, E, G, I, K, L, N, Q, R T, and V. In someembodiments, X is T. In some embodiments, an anti-Jagged1 antibodycomprises a heavy chain comprising the sequence of SEQ ID NO: 51, and alight chain comprising the sequence of SEQ ID NO: 53. In someembodiments, an anti-Jagged1 antibody comprises a heavy chain comprisingthe sequence of SEQ ID NO: 52, and a light chain comprising the sequenceof SEQ ID NO: 53. In some embodiments, an anti-Jagged1 antibodycomprises a heavy chain comprising the sequence of SEQ ID NO: 69, and alight chain comprising the sequence of SEQ ID NO: 75. In someembodiments, an anti-Jagged1 antibody comprises a heavy chain comprisingthe sequence of SEQ ID NO: 70, and a light chain comprising the sequenceof SEQ ID NO: 76.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-Jagged1 antibody provided herein. Forexample, in certain embodiments, an antibody is provided that binds tothe same epitope as an anti-Jagged1 antibody comprising a VH sequence ofSEQ ID NO: 33 and a VL sequence of SEQ ID NO: 34.

In a further aspect, the invention provides an antibody that competesfor binding with any of the antibodies provided herein.

In a further aspect of the invention, an anti-Jagged1 antibody accordingto any of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, ananti-Jagged1 antibody is an antibody fragment, e.g., a Fv, Fab, Fab′,scFv, diabody, or F(ab′)₂ fragment. In another embodiment, the antibodyis a full length antibody, e.g., an intact human IgG1 antibody or otherantibody class or isotype as defined herein.

In a further aspect, an anti-Jagged1 antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (MA). In one embodiment, an MA is performed with the Fab versionof an antibody of interest and its antigen. For example, solutionbinding affinity of Fabs for antigen is measured by equilibrating Fabwith a minimal concentration of (¹²⁵I)-labeled antigen in the presenceof a titration series of unlabeled antigen, then capturing bound antigenwith an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol.Biol. 293:865-881(1999)). To establish conditions for the assay,MICROTITER® multi-well plates (Thermo Scientific) are coated overnightwith 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mMsodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovineserum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (e.g., consistent with assessment of the anti-VEGF antibody,Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150μl/well of scintillant (MICROSCINT-20™; Packard) is added, and theplates are counted on a TOPCOUNT™ gamma counter (Packard) for tenminutes. Concentrations of each Fab that give less than or equal to 20%of maximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using a BIACORE® surfaceplasmon resonance assay. For example, an assay using a BIACORE®-2000 ora BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) is performed at 25° C.with immobilized antigen CMS chips at ˜10 response units (RU). In oneembodiment, carboxymethylated dextran biosensor chips (CMS, BIACORE,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for Jagged1 and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of Jagged1. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express Jagged1. Bispecific antibodiescan be prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to Jagged1 as wellas another, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or residues that contact antigen,with the resulting variant VH or VL being tested for binding affinity.Affinity maturation by constructing and reselecting from secondarylibraries has been described, e.g., in Hoogenboom et al. in Methods inMolecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa,N.J., (2001).) In some embodiments of affinity maturation, diversity isintroduced into the variable genes chosen for maturation by any of avariety of methods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). In some embodiments, an IgG1constant region comprising an N297G or N297A mutation substantiallylacks effector function. Examples of publications related to“defucosylated” or “fucose-deficient” antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in a animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-Jagged1 antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-Jagged1 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-Jagged1 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-Jagged1 antibodies provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with antibody A, A-1, A-2, or A-1(S101T) forbinding to human or murine Jagged1. In certain embodiments, such acompeting antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by A, A-1, A-2, or A-1(S101T).

Detailed exemplary methods for mapping an epitope to which an antibodybinds are provided in Morris (1996) “Epitope Mapping Protocols,” inMethods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized Jagged1 is incubated in asolution comprising a first labeled antibody that binds to Jagged1(e.g., A, A-1, A-2, or A-1(S101T)) and a second unlabeled antibody thatis being tested for its ability to compete with the first antibody forbinding to Jagged1. The second antibody may be present in a hybridomasupernatant. As a control, immobilized Jagged1 is incubated in asolution comprising the first labeled antibody but not the secondunlabeled antibody. After incubation under conditions permissive forbinding of the first antibody to Jagged1, excess unbound antibody isremoved, and the amount of label associated with immobilized Jagged1 ismeasured. If the amount of label associated with immobilized Jagged1 issubstantially reduced in the test sample relative to the control sample,then that indicates that the second antibody is competing with the firstantibody for binding to Jagged1. See Harlow and Lane (1988) Antibodies:A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-Jagged1antibodies thereof having biological activity. Biological activity mayinclude, e.g., inhibition of Jagged1-induced signaling through Notch1.In certain other embodiments, an antibody of the invention is tested forits ability to inhibit expression of a reporter gene that is responsiveto Jagged1-induced Notch signaling. Nonlimiting exemplary assays areprovided in the Examples. In certain embodiments, an antibody of theinvention is tested for such biological activity. Antibodies having suchbiological activity in vivo and/or in vitro are also provided.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-Jaggedantibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinyl sulfone)benzoate) which are commerciallyavailable (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-Jagged1 antibodies providedherein is useful for detecting the presence of Jagged1 in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue, such as cancerous tissues.

In one embodiment, an anti-Jagged1 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of Jagged1 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-Jagged1 antibody as described herein underconditions permissive for binding of the anti-Jagged1 antibody toJagged1, and detecting whether a complex is formed between theanti-Jagged1 antibody and Jagged1. Such method may be an in vitro or invivo method. In one embodiment, an anti-Jagged1 antibody is used toselect subjects eligible for therapy with an anti-Jagged1 antibody, e.g.where Jagged1 is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include cancer, e.g., breast cancer, lung cancer, braincancer, cervical cancer, colon cancer, liver cancer, bile duct cancer,pancreatic cancer, skin cancer, B-cell malignancies, and T-cellmalignancies.

In certain embodiments, labeled anti-Jagged1 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-Jagged1 antibody as describedherein are prepared by mixing such antibody having the desired degree ofpurity with one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, it may be desirable to further provide a cytotoxicagent, e.g., a chemotherapeutic agent. Such active ingredients aresuitably present in combination in amounts that are effective for thepurpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-Jagged1 antibodies provided herein may be used intherapeutic methods.

In one aspect, an anti-Jagged1 antibody for use as a medicament isprovided. In further aspects, an anti-Jagged1 antibody for use intreating a disease or disorder associated with aberrant Notch signaling,e.g. a cancer, is provided. In certain embodiments, an anti-Jagged1antibody for use in a method of treatment is provided. In certainembodiments, the invention provides an anti-Jagged1 antibody for use ina method of treating an individual having a cancer comprisingadministering to the individual an effective amount of the anti-Jagged1antibody. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent, e.g.,as described below.

In further embodiments, the invention provides an anti-Jagged1 antibodyfor use in inhibiting lung cancer growth. In certain embodiments, theinvention provides an anti-Jagged1 antibody for use in a method ofreducing lung cancer growth in an individual comprising administering tothe individual an effective of the anti-Jagged1 antibody to reducinglung cancer growth. In certain embodiments, the invention provides ananti-Jagged1 antibody for use in a method of reducing breast cancergrowth in an individual comprising administering to the individual aneffective of the anti-Jagged1 antibody to reducing breast cancer growth.An “individual” according to any of the above embodiments is preferablya human.

In some embodiments, an anti-Jagged1 antibody is provided for treatingallergy, asthma, autoimmune disease, diseases associated with gobletcell metaplasia (e.g., in lung) and/or excess mucus. Other allergicdiseases that may be treated with the anti-Jagged1 antibodies providedherein include, but are not limited to, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immune-mediated skindiseases include bullous skin diseases, erythema multiform and contactdermatitis; autoimmune disease include psoriasis, rheumatoid arthritis,juvenile chronic arthritis; inflammatory bowel disease (i.e., ulcerativecolitis, Crohn's disease); idiopathic interstitial pneumonia, diseasesassociated with goblet cell metaplasia (such as asthma, COPD, cysticfibrosis and Barrett's esophagus), lung diseases such as cysticfibrosis, gluten-sensitive enteropathy, and Whipple's disease;immunologic diseases of the lung such as eosinophilic pneumonia,idiopathic pulmonary fibrosis and hypersensitivity pneumonitis; chronicobstructive pulmonary disease, RSV infection, uvelitis, scleroderma,osteoporosis, and Hodgkin's lymphoma.

In a further aspect, the invention provides for the use of ananti-Jagged1 antibody in the manufacture or preparation of a medicament.In one embodiment, the medicament is for treatment of a disease ordisorder associated with aberrant Notch signaling. In one embodiment,the medicament is for treatment of a cancer. In a further embodiment,the medicament is for use in a method of treating a cancer comprisingadministering to an individual having a cancer an effective amount ofthe medicament. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below. An “individual”according to any of the above embodiments may be a human.

In a further aspect, the invention provides a method for treating adisease or disorder associated with aberrant Notch signaling. In oneembodiment, the method comprises administering to an individual havingsuch disease or disorder an effective amount of an anti-Jagged1antibody. In one embodiment, the method comprises administering to anindividual having a cancer an effective amount of an anti-Jagged1antibody. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, as described below. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent, asdescribed below. An “individual” according to any of the aboveembodiments may be a human.

In a further aspect, the invention provides a method for inhibitingcancer cell growth in an individual. In one embodiment, the methodcomprises administering to the individual an effective amount of ananti-Jagged1 antibody to inhibit cancer cell growth. In one embodiment,an “individual” is a human.

In some embodiments, the invention provides methods for treatingallergy, asthma, autoimmune disease, diseases associated with gobletcell metaplasia (e.g., in lung) and/or excess mucus in an individual. Insome embodiments, the invention provides methods for treating allergicrhinitis, atopic dermatitis, food hypersensitivity and urticaria;immune-mediated skin diseases include bullous skin diseases, erythemamultiform and contact dermatitis; autoimmune disease include psoriasis,rheumatoid arthritis, juvenile chronic arthritis; inflammatory boweldisease (i.e., ulcerative colitis, Crohn's disease); idiopathicinterstitial pneumonia, diseases associated with goblet cell metaplasia(such as asthma, COPD, cystic fibrosis and Barrett's esophagus), lungdiseases such as cystic fibrosis, gluten-sensitive enteropathy, andWhipple's disease; immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis; chronic obstructive pulmonary disease, RSV infection,uvelitis, scleroderma, osteoporosis, and/or Hodgkin's lymphoma in anindividual. In some embodiments, the method comprises administering tothe individual an effective amount of an anti-Jagged1 antibody providedherein. In some embodiments, an “individual” is a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-Jagged1 antibodies provided herein, e.g., foruse in any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the anti-Jagged1 antibodiesprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of theanti-Jagged1 antibodies provided herein and at least one additionaltherapeutic agent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is a cytotoxic agent. In certain embodiments, an additionaltherapeutic agent is an antibody.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent or agents. In one embodiment, administration of theanti-Jagged1 antibody and administration of an additional therapeuticagent occur within about one month, or within about one, two or threeweeks, or within about one, two, three, four, five, or six days, of eachother. Antibodies of the invention can also be used in combination withradiation therapy.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the antibody. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-Jagged1 antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-Jagged1 antibody.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1. Generation of Anti-Jagged Antibodies

a. Library Sorting and Screening to Identify Anti-Jagged1 Antibodies

Human phage antibody libraries with synthetic diversities in theselected complementarity determining regions, mimicking the naturaldiversity of human IgG repertoire, were used for panning Fab fragmentsdisplayed on the surface of M13 bacteriophage particles. HumanJag1-DSL-EGF1-4 (SEQ ID NO:6) or human Jag2-DSL-EGF1-4 (SEQ ID NO:8) wasused as antigen for library sorting. Nunc 96 well Maxisorp immunoplateswere coated overnight at 4° C. with target antigen (10 μg/ml) and wereblocked for 1 hour at room temperature with phage blocking buffer PBST(phosphate-buffered saline (PBS) and 1% (w/v) bovine serum albumin (BSA)and 0.05% (v/v) tween-20). Antibody phage libraries VH (see, e.g., Leeet al., J. Immunol. Meth. 284:119-132 (2004)) and VH/VL (see Liang etal., JMB. 366: 815-829 (2007)) were added to antigen plates separatelyand incubated overnight at room temperature. The following dayantigen-coated plates were washed ten times with PBT (PBS with 0.05%Tween-20), and bound phage were eluted with 50 mM HCl and 500 mM NaClfor 30 minutes and neutralized with an equal volume of 1 M Tris base(pH7.5). Recovered phages were amplified in E. coli XL-1 Blue cells.During the subsequent selection rounds, incubation of antibody phagewith the antigen-coated plates was reduced to 2-3 hours, and thestringency of plate washing was gradually increased.

After 4 rounds of panning, significant enrichment was observed. 96clones were picked each from VH and VH/VL library sorting to determinewhether they specifically bound to human Jagged1 or Jagged2. Thevariable regions of these clones were PCR sequenced to identify uniquesequence clones. The affinities of phage antibodies were ranked usingspot competition ELISA. The phage antibody IC50 values were furtherdetermined using competitive phage-binding ELISA. Unique phageantibodies that bind specifically to human Jagged1 (and not Jagged2),Jagged2 (and not Jagged1), or to both Jagged1 and Jagged2 were chosenand reformatted to full-length IgGs for evaluation in in vitro cellassays.

Clones of interest were reformatted into IgGs by cloning V_(L) and V_(H)regions of individual clones into a pRK mammalian cell expression vector(pRK.LPG3.HumanKappa) containing the human kappa constant domain, andexpression vector (pRK.LPG4.HumanHC) encoding the full-length human IgG1constant domain, respectively (Shields et al., J. Biol Chem 2000; 276:6591-6604). The antibodies were then transiently expressed in mammalianCHO cells, and purified with a protein A column.

b. Construction of Libraries for Affinity Improvement of Clones Derivedfrom the V_(H) or V_(H)V_(L) Libraries

Phagemid pW0703, derived from phagemid pV0350-2b (Lee et al., J. Mol.Biol 340, 1073-1093 (2004), containing stop codon (TAA) in all CDR-L3positions and displaying monovalent Fab on the surface of M13bacteriophage) served as the library templates for grafting heavy chainvariable domains (V_(H)) of clones of interest from the V_(H) libraryfor affinity maturation. Both hard and soft randomization strategieswere used for affinity maturation. For hard randomization, one lightchain library with selected positions of the three light chain CDRs wasrandomized using amino acids designed to mimic natural human antibodiesand the designed DNA degeneracy was as described in Lee et al. (J. Mol.Biol 340, 1073-1093 (2004)). To achieve the soft randomizationconditions, which introduced the mutation rate of approximately 50% atthe selected positions, the mutagenic DNA was synthesized with70-10-10-10 mixtures of bases favoring the wild type nucleotides (Gallopet al., Journal of Medicinal Chemistry 37:1233-1251 (1994)). For softrandomization, residues at positions 91-96 of CDR-L3, 30-33, 35 ofCDR-H1, 50, 52, 53-54, and 56 of CDR-H2, 95-98 of CDR-H3 were targeted;and three different combinations of CDR loops, H1/L3, H2/L3, and H3/L3,were selected for randomization.

For clones originated from V_(H)V_(L) library, phagemids containing 4stop codons (TAA) in each CDR and displaying monovalent Fab on thesurface of M13 bacteriophage were generated individually, and served asthe templates for kunkel mutagenesis for the construction of affinitymaturation libraries. Only soft randomization strategy was used forclones derived from V_(H)V_(L) library, as diversity of CDR-L3 was builtinto the naïve library. To achieve the soft randomization conditions,residues at positions 28-31 of CDR-L1, 50, 53-55 of CDR-L2, 91-96 ofCDR-L3, 30-35 of CDR-H1, 50-56 of CDR-H2, 95-100 of CDR-H3 weretargeted; and four different combinations of CDR loops, H1/L3*, H2/L3*,and H3/L3* and L1/L2/L3* (where * denotes the position of stop codons onthe template), were selected for randomization.

c. Phage Sorting Strategy to Generate Affinity Improvement

For affinity improvement selection, Jag1 or Jag2 antigens were firstbiotinylated under limiting reagent condition. Phage libraries weresubjected to one round of plate sorting and five rounds of solutionsorting with increasing stringency. For the first round of platesorting, 10 ug/ml antigen was first coated on Maxisorp plate andpreblocked with blocking buffer (1% BSA and 0.05% Tween20 in PBS). 3O.D./ml in blocking buffer of phage input were incubated to antigenplates for 3 hours. The wells were washed with PBS-0.05% Tween20 tentimes. Bound phage was eluted with 150 μl/well 50 mM HCl, 500 mM KCl for30 minutes, and subsequently neutralized by 50 μl/well of 1M Tris pH8,titered, and propagated for the next round. For subsequent rounds,panning of the phage libraries was done in solution phase, where phagelibrary was incubated with 100 nM biotinylated target protein (theconcentration is based on parental clone phage IC50 value) in 100 μlbuffer containing 1% Superblock (Pierce Biotechnology) and 0.05% Tween20for 2 hours at room temperature. The mixture was further diluted 10×with 1% Superblock, and 100 μl/well was applied to neutravidin-coatedwells (10 μg/ml) for 30 minutes at room temperature with gentle shaking.To determine background binding, control wells containing phage werecaptured on neutravidin-coated plates. Bound phage was then washed,eluted and propagated as described for first round. Five more rounds ofsolution sorting were carried out together with increasing selectionstringency. The first couple rounds of which is for on-rate selection bydecreasing biotinylated target protein concentration from 100 nM to 0.1nM, and the last two rounds of which is for off-rate selection by addingexcess amounts of non-biotinylated target protein (300 to 1000 foldmore) to compete off weaker binders at room temperature.

d. High Throughput Affinity Screening ELISA (Single Spot Competition)

Colonies were picked from the sixth round of screening. Colonies weregrown overnight at 37° C. in 150 μl/well of 2YT media with 50 μg/mlcarbenicillin and 1×10¹⁰/ml M13KO7 in 96-well plate (Falcon). From thesame plate, a colony of XL-1 infected parental phage was picked ascontrol. 96-well Nunc Maxisorp plates were coated with 100 μl/well ofeither Jag1 or Jag2 (0.5 μg/ml) in PBS at 4° C. overnight. The plateswere blocked with 150 μl of 1% BSA and 0.05% Tween20 in PBS 20 for 1hour.

35 μl of the phage supernatant was diluted with to 75 μl of in ELISA(enzyme linked immunosorbent assay) buffer (PBS with 0.5% BSA, 0.05%Tween20) with or without 5 nM Jag1 or Jag2 and let incubate for 1 hourat room temperature in an F plate (NUNC). 95 μl of mixture wastransferred side by side to the antigen coated plates. The plate wasgently shaken for 15 min and was washed ten times with PBS-0.05% Tween20. The binding was quantified by adding horseradish peroxidase(HRP)-conjugated anti-M13 antibody in ELISA buffer (1:2500) andincubated for 30 minutes at room temperature. The plates were washedwith PBS-0.05% Tween 20 ten times. Next, 100 μl/well of Peroxidasesubstrate was added to the well and incubated for 5 minutes at roomtemperature. The reaction was stopped by adding 100 μl 0.1 M PhosphoricAcid (H₃PO₄) to each well and allowed to incubate for 5 minutes at roomtemperature. The O.D. (optical density) of the yellow color in each wellwas determined using a standard ELISA plate reader at 450 nm. Incomparison to the OD_(450nm) reduction (%) of the well of parental phage(100%), clones that had the OD_(450nm) reduction (%) lower than 50% werepicked for sequence analysis. Unique clones were selected for phagepreparation to determine binding affinity (phage IC50) against eitherJag1 or Jag2 by comparison to respective parental clones. Then the mostaffinity-improved clones were reformatted into human IgG1 for antibodyproduction and further BIAcore binding kinetic analysis and other invitro or in vivo assay.

Further screening rounds identified antibodies specific for only one ofthe Jagged family members, as determined by ELISA. Antibody A boundhuman and murine Jagged1, but not Jagged2 (FIG. 8; heavy chain variableregion sequence shown in SEQ ID NO: 9, light chain variable regionsequence shown in SEQ ID NO: 10). Conversely, antibody B (the parentalantibody of antibody B-3) bound human and murine Jagged2, but notJagged1 (FIG. 8). C-1 binds to both Jagged1 and Jagged2, and served as acontrol. The heavy chain and light chain variable region sequences forantibody C-1 are shown in FIG. 4.

Example 2. Antibody Binding Affinities and Epitope Mapping

Binding affinities of anti-Jagged1 phage antibodies were measured bySurface Plasmon Resonance (SRP) using a BIAcore™-3000 instrument.Anti-Jagged1/2 phage human IgGs were captured by mouse anti-human IgGcoated on the CMS sensor chip to achieve approximately 150 responseunits (RU). For kinetics measurements, two-fold serial dilutions ofhuman or mouse Jag1/2 DSL EGF1-4 (1.95 nM to 250 nM) were injected inPBT buffer (PBS with 0.05% Tween 20) at 25° C. with a flow rate of 30ml/min. Association rates (k_(on)) and dissociation rates (k_(off)) werecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2). The equilibrium dissociation constant(k_(d)) was calculated as the ratio k_(off)/k_(on).

Table 2 summarizes the binding constants for antibodies A, A-1, A-2, B,and B-3 binding to purified human Jagged1, human Jagged2, and mouseJagged2. Parent antibody A specifically bound to human and murineJagged1. The affinity matured antibodies A-1 and A-2 bound both humanand murine Jagged1 with high affinity. Antibodies A, A-1 and A-2 did notbind human or murine Jagged2. Conversely, antibodies B and B-3 did notbind human or murine Jagged1. B-3 specifically bound to human and mouseJagged2.

TABLE 2 Biacore summary table Human Jag1 Human Jag2 Mouse Jag2 kon koffKd kon koff Kd kon koff Kd Ab (1/Ms) (1/s) (M) (1/Ms) (1/s) (M) (1/Ms)(1/s) (M) A 2.3E+04 2.1E−03 9.4E−08 No binding up to 0.5 μM A-1 8.3E+045.9E−05 7.1E−10 A-2 2.3E+05 7.1E−05 3.0E−10 B No binding up to 0.5 μM2.5E+06 2.6E−03 1.0E−09 2.5E+06  2.6E−03 1.0E−09 B-3 5.8E+05 1.75E−043.0E−10

The heavy chain and light chain variable region sequences for antibody Aare shown in SEQ ID NOs: 9 and 10, respectively. The heavy chain andlight chain variable region sequences for antibody A-1 are shown in SEQID NOs: 17 and 18, respectively. The heavy chain and light chainvariable region sequences for antibody A-2 are shown in SEQ ID NOs: 25and 26, respectively. The heavy chain and light chain variable regionsequences for antibody B-3 are shown in SEQ ID NOs: 41 and 42,respectively. The heavy chain and light chain variable region sequencesfor antibody B are shown in FIG. 4.

Example 3. Anti-Jagged Antagonist Antibodies Inhibit Jagged1-InducedSignaling In Vitro

To determine whether anti-Jagged antibodies can act as antagonists ofJagged-induced Notch signaling, co-culture experiments were performedessentially as described by Wu et al., Nature 464, 1052-1057 (15 Apr.2010). NIH-3T3 cells engineered to express Jagged1, as the Notch ligand,were co-cultured with NIH 3T3 cells that stably express Notch1 and thatwere transiently transfected to express a Notch-responsive TP-1 (12×CSL)firefly luciferase reporter and a constitutively expressed Renillaluciferase reporter (pRL-CMV, Promega). Strong Notch reporter signal(Firefly luciferase) was observed in the co-culture (FIG. 12, J1induced-Positive Control). Reporter expression was reduced to backgroundlevels when a γ-secretase inhibitor was added to the co-culture,demonstrating Notch-dependent expression of the reporter construct. Datanot shown.

Addition of increasing amounts (0.016-50 μg/ml) of anti-Jagged antibodyA-2 (heavy chain and light chain variable region sequences of SEQ IDNOs: 25 and 26, respectively) or B-3 (heavy chain and light chainvariable region sequences of SEQ ID NOs: 41 and 42, respectively)resulted in dose-dependent inhibition of reporter expression (FIG. 9).Signaling was induced by Jagged1 (FIG. 9A, dark gray columns) or byJagged2 (FIG. 9B, light gray columns) and inhibition was determined asdescribed above. Controls included cultures that were not stimulatedwith ligand and not treated with antibody (FIGS. 9A and B, Untreated),not stimulated with ligand (FIGS. 9A and B, No Stimulation), treatedwith 5-10 μg/ml isotype control antibody (FIGS. 9A and B, agD),stimulated with ligand but not treated with antibody (FIGS. 9A and B,Stim/no AB), treated with 5 μM of the gamma-secretase inhibitor DAPT orthe DAPT vehicle control of DMSO.

Antibody A-2 inhibited Jagged1-induced signaling, but notJagged2-induced signaling, in a dose-dependent manner (FIG. 9A). TheIC₅₀ for A-2 was between 2 and 10 μg/ml for Jagged1 inhibition whereaslittle or no Jagged2 inhibition was observed even at the highestconcentration of 50 μg/ml. The results demonstrate that antibody A-2 isa Jagged1-selective antagonist, i.e., antibody A-2 inhibitsJagged1-mediated signaling, but not Jagged2-mediated signaling. Incontrast, antibody B-3 potently inhibited Jagged2-induced signaling atthe lowest concentration tested but did not inhibit Jagged1-inducedsignaling at the highest concentration tested, thus establishing B-3 asa Jagged1-selective antagonist (FIG. 9B).

Example 4. Effect of Anti-Jagged Antibody Treatment on Body Weight

As described above, gamma-secretase inhibitors, and other inhibitors ofmultiple Notch receptors, cause weight loss and intestinal goblet cellmetaplasia, which is undesirable for clinical administration. Todetermine how the antibodies described herein affect body weight andintestinal health, mice were dosed twice per week with the anti-Jagged1antibody A-2 (5-20 mpk; heavy chain and light chain variable regionsequences of SEQ ID NOs: 25 and 26, respectively), the anti-Jagged2antibody B-3 (5-20 mpk; heavy chain and light chain variable regionsequences of SEQ ID NOs: 41 and 42, respectively), the antibody A-2 andB-3 together (5 mpk each), an anti-Jagged1/2 antibody that binds to bothJagged 1 and Jagged 2 (C-1; 5-10 mg antibody per kg mouse body weight(mpk); heavy and light chain variable regions sequence shown in FIG. 4),or the isotype control anti-gD antibody (20 mpk). The isotype controlantibody was also used to bring the total antibody concentration of eachdosing to 20 mpk. Total body weight of each mouse was determined priorto first administration of antibodies and monitored until day 12 of thestudy. The average body weight changes are depicted in FIG. 10, graphedas a percentage of starting body weight. Dual inhibition of Jagged1 andJagged2, using either the anti-Jagged1/2 antibody C-1 or a combinationof the Jagged1-specific antibody A-2 and the Jagged2-specific antibodyB-3 together, caused rapid and substantial weight loss (FIG. 10A). Byday 4, some mice that received the anti-Jagged1/2 antibody C-1 had lostover 5% of their bodyweight, which progressed to nearly 8-10% loss inbody weight by day 7 (FIG. 10A). Mice that received both A-2 and B-3also lost weight rapidly, in some cases up to 17% by day 11 (FIG. 10A).In contrast, none of the Jagged1-specific or Jagged2-specific antibodiesalone caused weight loss over the course of the study at either 5 or 20mpk (FIG. 10A). Treatment with the combination of anti-Jagged1 plusanti-Jagged2 antibodies resulted in decreased food intake (FIG. 10B),which correlated with the observed decrease in body weight (FIG. 10A)and suggested that decreased food intake could partly or entirelyaccount for the correlated body weight decreases.

Example 5. Anti-Jagged1 Antagonist Antibodies Inhibit Human Lung CancerCell Growth In Vivo

Harlan athymic nude mice were inoculated subcutaneously with Calu-6cells, a human non-small cell lung cancer line. After tumor volumereached approximately 200 cubic mm, mice were injected intraperitoneally(IP) twice per week (days 0, 4, 7, 11, 14 and 18) with 20 mpk of eitheranti-gD isotype control antibody (n=10) or with anti-Jagged1 antibodyA-2 (n=10; heavy chain and light chain variable region sequences of SEQID NOs: 25 and 26, respectively). Tumor volume in each mouse wasmeasured with calipers for another 19 days. Total body weight of eachmouse was monitored over the course of the study.

Tumors in mice treated with anti-Jagged1 showed a significant decreasein tumor volume relative to tumors in the control group (FIG. 11A). Theeffect of the anti-Jagged1 antibody treatment could be detected as earlyas day seven after treatment (FIG. 11A). At day 18, the average tumorvolume in mice that received the anti-Jagged1 antibody reachedapproximately 500 mm³, while average tumor volume in control animalsreached approximately 750 mm³ at day 18. No significant change in bodyweight between the treatment and control group could be observed (FIG.11B).

Example 6. Anti-Jagged1 and Anti-Jagged2 Antibodies Inhibit Human BreastCancer Cell Growth In Vivo

C.B-17 SCID.bg mice were inoculated in the mammary fat pad withMDA-MD-468 cells, a human basal breast cancer line. After tumor volumereached approximately 200 cubic mm, mice were dosed IP with 30 mpk ofeither anti-gD isotype control antibody (human IgG1 isotype),anti-ragweed isotype control antibody (murine IgG2a isotype),anti-Jagged1 antibody A-2 (heavy chain and light chain variable regionsequences of SEQ ID NOs: 25 and 26, respectively) in the human IgG1backbone, anti-Jagged1 antibody A-2 in the murine IgG2a backbone oranti-Jagged2 antibody B-3 (heavy chain and light chain variable regionsequences of SEQ ID NOs: 41 and 42, respectively) in the human IgG1backbone on days 0, 4, 7, 12, 15, 18, 22, 25, 29, 32, 36, 43, 50, and57. Tumor volume (y-axis) was measured with calipers for 60 days afterthe first injection. The tumor volumes for each group (n=9 per group)were plotted using a linear mixed effects model (FIG. 12A). Tumorvolumes for each mouse in each group are depicted in FIG. 12B.

Example 7: Anti-Jagged1 Antibodies are Cleaved in the Heavy Chain

Antibodies A (heavy chain and light chain variable region sequences ofSEQ ID NOs: 9 and 10, respectively), A-1 (heavy chain and light chainvariable region sequences of SEQ ID NOs: 17 and 18, respectively), andA-2 (heavy chain and light chain variable region sequences of SEQ IDNOs: 25 and 26, respectively) were analyzed by SDS-PAGE and massspectrometry for integrity of the heavy and light chains. For SDS-PAGEanalysis, each antibody sample was mixed in a 1:1 v/v ratio with 2×Tris-Glycine SDS Sample Buffer (Novex LC2676), in the absence andpresence of 10 mM DTT. Samples were heated at 95° C. for 5 minutes, and2 μg of each sample was loaded onto a Novex 4-20% SDS-PAGE, 1.0 mm gel(Novex EC6025). 10 μL of Mark 12 molecular weight standard (Invitrogen100006637) was also loaded onto the gel. Electrophoresis was run in 1×Tris-Glycine SDS Running Buffer (Invitrogen LC2675-5) at a constant 250Vuntil the tracking dye reached the bottom of the gel. The gel was thenstained with a Coommassie-based stain (Expedeon InstantBlue #ISB1L).

For mass spectrometry analysis, each antibody was diluted to a finalconcentration of 1 mg/mL in PBS. The pH of the antibody was increased to8.0 with addition of 1:10 v/v of 1.0M Tris, pH 8.0. DTT to a finalconcentration of 10 mM in solution was added to reduce the antibody. Thesample was then heated at 37° C. for 15 minutes. Samples were theninjected onto a PLRP-S 1000 Å, 8 μm, 2.1×50 mm column (Agilent,PL1912-1802) heated to 80° C. using an Agilent 1200 HPLC system,followed by electrospray ionization on an Agilent 6210 TOF LC/MS system.

The results of those analyses are shown in FIG. 13. The SDS-PAGEanalysis (FIG. 13A) revealed that a fraction of the heavy chain (HC) iscleaved in each of the antibodies. The bands corresponding to the intactHC and light chain (LC), as well as the carboxy (C)-terminal and amino(N)-terminal cleaved fragments of the HC, are marked to the right of thegel. A representative mass spectrometry analysis, for antibody A-1, isshown in FIG. 13B. This analysis indicated that the cleavage site wasbetween HC amino acids G100 and S101 (sequential numbering,corresponding to G96 and S97 according to Kabat numbering) in CDR3, asdiagramed in the HC amino acid sequence in FIG. 13C, with the arrowmarking the cleavage position. Similar analyses revealed that the HCcleavage occurred in all tested preparations of these antibodies(including following expression in two different cell types, CHO and293) and that cleavage occurred independent of the type of antibody Fcregion (human IgG1 or murine IgG2a).

An SDS-PAGE gel of the anti-Jagged1 antibodies was run substantially asdescribed above. Rather than staining with Coomassie-based stain,antibody was transferred onto PVDF membrane (Invitrogen LC2002) usingXCell Blot Module (Invitrogen EI9051) at constant 0.35A. The membranewas stained with Coomassie Blue R-250. The samples on membrane weresubjected to N-terminal sequence analysis using the Applied BiosystemsProcise Sequencer 494 according to the sequencing principle described inNiall, 1973, Meth. Enzymol. 27: 942-1010.

The results of the sequencing analysis are shown in FIG. 14. This methodconfirmed that the cleavage site was the same one predicted from themass spectrometry results, which is between G100 and S101 (sequentialnumbering, corresponding to G96 and S97 according to Kabat numbering) ofthe HC sequence.

The heavy chain cleavage of anti-Jagged1 antibodies A, A-1, And A-2 wasunexpected. An analysis of the amino acid sequence surrounding thecleavage site identified no known protease cleavage sites. The mechanismof the cleavage is unclear, and is not readily apparent from thesequences of the antibodies.

Example 8: Mutation of Heavy Chain S101 Reduces Cleavage of Anti-Jagged1Antibody Heavy Chain

Since the observed cleavage of the anti-Jagged1 antibodies wasunexpected and the mechanism unclear, it was not known whether changesto the antibody sequence could prevent the cleavage, while retaining theaffinity and efficacy of the antibody. In addition, it was not knownwhat position(s) in the antibody sequence should be changed to preventcleavage. To determine whether antibody cleavage could be prevented bychanging the heavy chain sequence, a series of amino acid changes weremade at heavy chain position S101 (sequential numbering of the heavychain variable region sequence of SEQ ID NO: 17, corresponding to S97according to Kabat numbering). The antibodies were expressed inmammalian cells and purified according to standard procedures. Cleavagewas analyzed by SDS-PAGE as described in Example 7. The results areshown in FIG. 15. The amino acid changes at position S101 significantlyreduced or eliminated HC cleavage, although some cleavage was detectedwith the S101H mutation (FIG. 15, lane 5). These results were confirmedby mass spectrometry, performed as described in Example 7.

To determine the effects of the changes on binding to a purified Jag1extracellular domain protein fragment, mutant A-1 antibody bindingaffinities were measured using BIAcore. Table 3 shows a summary of thefragmentation and Jagged1 binding affinities for each of the mutant A-1antibodies.

TABLE 3 Fragmentation and Jagged1 binding affinities of mutant A-1antibodies HC Fragmentation Detected? Mass Jag 1 Binding (BIAcore) mAbVariants Spec SDS-PAGE ka (1/Ms) kd (1/s) KD (M) A-1 yes yes 1.80E+051.44E−04 7.99E−10 G100, S101 (WT) A-1 yes yes G100A, S101 A-1 no no1.46E+05 6.75E−04 4.62E−09 G100, S101A A-1 no no 1.02E+05 5.56E−045.47E−09 G100, S101D A-1 no no 1.67E+05 1.15E−03 6.88E−09 G100, S101EA-1 no no 1.54E+05 8.72E−04 5.67E−09 G100, S101G A-1 yes yes 1.27E+055.62E−04 4.43E−09 G100, S101H A-1 no no 9.88E+04 5.90E−04 5.97E−09 G100,S101I A-1 no no 1.18E+05 8.43E−04 7.16E−09 G100, S101K A-1 no no8.34E+04 6.49E−04 7.79E−09 G100, S101L A-1 no no 1.36E+05 7.97E−045.87E−09 G100, S101N A-1 no no 1.03E+05 5.82E−04 5.64E−09 G100, S101QA-1 no no 1.02E+05 6.90E−04 6.75E−09 G100, S101R A-1 no no 1.31E+053.41E−04 2.61E−09 G100, S101T A-1 no no 1.20E+05 5.80E−04 4.84E−09 G100,S101VAs shown in Table 3, the changes to the amino acid residue at position101 reduced cleavage to undetectable levels, except for S101H. Inaddition, since the mechanism of cleavage was unknown, a change toposition 100, G100A, was also tested. The G100A mutant was stillcleaved. See Table 3. Surprisingly, given that the mutations were madein HVR-H3, the S101 mutant antibodies retained the ability to bindJagged1.

Example 9: Effect of Temperature and Freeze-Thaw Cycling on Cleavage ofAnti-Jagged1 Antibody Heavy Chain

To assess whether anti-Jagged1 antibody cleavage is increased byincubation at increasing temperatures, various independent preparationsof anti-Jagged1 antibodies A-1 (heavy chain and light chain variableregion sequences of SEQ ID NOs: 17 and 18, respectively) and A-2 (heavychain and light chain variable region sequences of SEQ ID NOs: 25 and26, respectively), as well as an isotype control antibody (notanti-Jagged1), were incubated for 10 min at 70° C. or 95° C. The extentof cleavage was assessed using standard SDS-PAGE and protein staining.The results are shown in FIG. 16. Each of the anti-Jagged1 antibodypreparations was cleaved at 70° C., while the control antibody was not(FIG. 16A). As summarized in the table (FIG. 16B), the extent ofanti-Jagged1 cleavage was not significantly or consistently altered byincubation at 95° C. versus 70° C.

To assess whether cleavage resulted from freeze-thaw cycling ofanti-Jagged1 antibody preparations, antibody A-1 (heavy chain and lightchain variable region sequences of SEQ ID NOs: 17 and 18, respectively)was subjected to multiple rounds of freezing at −80° C. followed bythawing. 2 μg of each sample was then analyzed by SDS-PAGE and proteinstaining under non-reducing (−DTT) or reducing (+DTT) conditions, asindicated. The stained gel was imaged using a Biorad GelDoc Easy Imagerinstrument and densitometry analysis was performed using Biorad ImageLab software. The results of that experiment are shown in FIG. 17. F/T 1refers to the original sample, with increasing numbers indicating thenumber of additional rounds of freeze-thaw cycles. For each cycle, theA-1 antibody was frozen at −80° C. and then thawed at room temperature.An aliquot was removed for SDS-PAGE. The freeze/thaw was repeated twicemore for a total of 3 freeze/thaw cycles, with aliquots being removedafter each thaw step. The table (FIG. 17B) summarizes the percentage ofcleavage under each condition. The experiment revealed that additionalrounds of freeze-thaw had little impact on the percentage of cleavage.

Example 10: Jagged1 Blocking Activity of A-1 and A-1(S101T) In Vitro

In vitro co-culture assays of Jag1-induced Notch reporter activity wereperformed to measure the Jag1 blocking activity of A-1 (heavy chain andlight chain variable region sequences of SEQ ID NOs: 17 and 18,respectively) and A-1(S101T) (heavy chain and light chain variableregion sequences of SEQ ID NOs: 33 and 34, respectively). U87MG cells,which express high levels of Notch2, were co-transfected with aNotch-responsive TP-1 (12×CSL) Firefly (FF) luciferase reporter and aconstitutively expressed Renilla luciferase reporter (pRL-CMV, Promega)to control for transfection efficiency. See Wu et al., 2010, Nature 464:1052-1057. Anti-Jagged1 antibody A-1 or A-1(S101T), isotype controlantibody, 5 μM DAPT (Calbiochem), or DMSO vehicle control was added withligand-expressing cells (NIH-3T3 cells stably transfected with humanJag1 or a no ligand control) 6 hours after transfection. Luciferaseactivities were measured after 20 hours of co-culture (Promega, Dual GloLuciferase). Typically, four replicates were analyzed for eachcondition, and values were expressed as relative luciferase units(Firefly signal divided by the Renilla signal) and graphed as apercentage of Jag1-induced activity per anti-Ragweed control.

The results of that experiment are shown in FIG. 18. FF and Renillaluciferase measurements were taken (FIGS. 18B and C, respectively)according to standard methods, and the FF to Renilla luciferase ratioswere graphed as a normalized measurement of Jag1-induced Notch activity(FIG. 18A). The results show that A-1 and A-1(S101T) both inhibitJag1-induced Notch signaling in a dose-dependent manner. See FIGS. 18Aand B. Thus, A-1(S101T) retains the anti-Jagged1 blocking activity ofparental antibody A-1.

Example 11: Jagged1 Blocking Activity of A-1 and A-1(S101T) In Vivo

To assess Jagged1 blocking activity of anti-Jagged1 antibodies in vivo,the club and ciliated cell composition of mouse lung bronchiolarepithelium was measured after dosing mice with control or anti-Jagged1antibodies. Wild-type, eight-week old BALB/c female mice (three mice pergroup) were dosed on day 0 as follows (all groups contained anti-Jagged2antibody (B-3) to sensitize the bronchiolar epithelium to reveal clearlymeasurable effects of anti-Jagged1 activity):

-   -   1. Control 1×—isotype control (15 mg antibody per kg mouse body        weight)+anti-Jag2 B-3 (15 mg/kg; heavy chain and light chain        variable region sequences of SEQ ID NOs: 41 and 42,        respectively)    -   2. A-1 1×—anti-Jag1 A-1 (15 mg/kg; heavy chain and light chain        variable region sequences of SEQ ID NOs: 17 and 18,        respectively)+anti-Jag2 B-3 (15 mg/kg)    -   3. A-1 0.5×—anti-Jag1 A-1 (7.5 mg/kg)+anti-Jag2 B-3 (15 mg/kg)    -   4. A-1 0.25×—anti-Jag1 A-1 (3.75 mg/kg)+anti-Jag2 B-3 (15 mg/kg)    -   5. A-1-S101T 2×—anti-Jag1 A-1(S101T) (30 mg/kg; heavy chain and        light chain variable region sequences of SEQ ID NOs: 33 and 34,        respectively)+anti-Jag2 B-3 (15 mg/kg)    -   6. A-1-S101T 1×—anti-Jag1 A-1(S101T) (15 mg/kg)+anti-Jag2 B-3        (15 mg/kg)    -   7. A-1-S101T 0.5×—anti-Jag1 A-1(S101T) (7.5 mg/kg)+anti-Jag2 B-3        (15 mg/kg)    -   8. A-1-S101T 0.25×—anti-Jag1 A-1(S101T) (3.75 mg/kg)+anti-Jag2        B-3 (15 mg/kg)

On day 5, lungs were harvested, inflated, fixed and stained forimmunofluorescence (IF) as follows. Lungs were inflated with 4% PFA inPBS. The entire lung was transferred to 10% neutral buffered formalin(NBF) and fixed over-night at room temperature. Fixed lungs weretransferred to 70% ethanol for at least 24 hours. The lungs wereparaffin embedded and sectioned at 5 μm. Immunofluorescence staining forciliated and Clara cells was as follows. Slides were de-paraffinized andantigens were retrieved by boiling the slides in citrate buffer (Dako51700) in a pressure cooker for 15 minutes at 125° C. Slides werebriefly rinsed in 2×PBS, and then permeablized with 0.2% Triton-X100 inPBS for 45 minutes or 3×15 minutes. Sections were blocked with 5% FBS/2%BSA for 1 hour. Slides were then incubated with goat anti-CC10 (1:1000)and mouse anti-acetylated alpha tubulin (1:200) in blocking buffer for 2to 3 hours or overnight. Slides were washed 3 times for 15 minutes withPBS. Slides were incubated with secondary antibodies for 1 hour(Invitrogen Alexa Fluor secondary antibodies diluted 1:1000), thenrinsed twice for 15 minutes with PBS. Nuclei were stained with DAPI (0.5ug/ml) for 15 minutes. Slides were then rinsed twice for 15 minutes withPBS and coverslipped.

The results of that experiment are shown in FIG. 19. Blocking Jagged1and Jagged2 signaling causes an increase in the number of ciliated cells(as marked by immunofluorescent detection of alpha-tubulin in red) and adecrease in the number of club cells (as marked by immunofluorescentdetection of CC10 in green) in the mouse bronchiolar epithelium.Blocking both Jagged1 plus Jagged2 results in a near complete loss ofclub cells, such that the resulting epithelium consists primarily ofciliated cells (red; see “A-1, 1×” and “A-1-S101T, 2×” in FIG. 19). A-1and A-1(S101T) both inhibited Jagged1-induced Notch signaling in vivo ina dose-dependent manner. Thus, A-1(S101T) retains the anti-Jagged1blocking activity of parental antibody A-1 in vivo.

Example 12: Anti-Jagged1 Antibodies A-1 and A-1(S101T) Inhibit Growth ofLiver Cancer Tumors In Vivo

A human patient-derived liver cancer tumor, LIV#78 (Genendesign, China),was grown as a subcutaneous xenograft in BALB-c nude immunocompromisedmice. When tumors grew to between 150 to 200 mm³, the mice were groupedinto seven treatment groups with ten mice per group and dosed once perweek (except for group 5, which was dosed once every three weeks) at theindicated dose (in mg of antibody per kg of mouse body weight) of theindicated antibody (A-1(S101T) antibody has the heavy chain and lightchain sequences of SEQ ID NOs: 51 and 53, respectively;“A-1-DANG(effectorless)” has the heavy and light chain sequences of SEQID NOs: 52 and 53, respectively; and A-1 antibody has the heavy chainand light chain sequences of SEQ ID NOs: 81 and 53, respectively). SeeFIG. 20B. Tumor volumes were assessed by caliper measurements(length×width×height/2).

FIG. 20A shows a LME (linear mixed effects) graph of tumor volume ineach treatment group over the course of the study. FIG. 20B summarizesthe growth statistics, group identities and dosing regimens. Theanti-Jagged1 A-1 and A-1(S101T) antibodies significantly inhibited livercancer growth in vivo; multiple PRs (partial responses) were observedwith each of the anti-Jagged1 antibodies. Likewise, none of theanti-Jagged1 treated groups showed a doubling of tumor volume during the44 days of the study, whereas the control group showed a time toprogression of tumor doubling (TTP 2×) of 18.5 days. See FIG. 20B. Thepercentage of tumor growth inhibition (% TGI, where “lower” and “upper”refer to the lowest and uppermost % TGI measurements, respectively, forindividual animals in each group) as a function of area under the curveper day (AUC/day) compared to the control group depended on the dose ofanti-Jagged1 A-1-S101T, consistent with tumor growth inhibitionreflecting the extent of Jagged1 inhibition. Likewise, inhibition oftumor growth was similar using A-1 or A-1(S101T). Compare, e.g., groups3 and 7, FIG. 20B. Tumor growth inhibition did not depend on antibodyeffector function because a heavy chain N297G mutant form of A-1, whichlacks effector function, inhibited tumor growth as effectively as A-1.Compare groups 6 and 7, FIG. 20B.

FIG. 21A shows a LME (linear mixed effects) graph of mouse body weightover time for the mice shown in FIG. 20. FIG. 20B shows various bodyweight parameters for the treatment groups, including the % change inbody weight on the last day of the study (% BW Last Day), the maximum %change in body weight (Max % BW), and the day on which the maximumchange in bodyweight occurred (Max % BW Day), and the (AUC/Day(lower,upper)). The body weight graphs for the treatment groups,including the control group, are statistically indistinguishable,indicating that anti-Jagged1 treatment was well-tolerated.

Example 13: Blocking Jagged1 Inhibits Goblet Cell Metaplasia In Vivo

Following a 35 day period of sensitization to intraperitonealy injectedovalbumin, mice were challenged with aerosolized ovalbumin for 7consecutive days, after which they were sacrificed and analyzed for thenumber of goblet cells. Mice were treated with control, anti-Jagged1 A-2antibody (with murine IgG2a Fc), anti-Jagged2 B-3 antibody (with murineIgG2a Fc) or the combination of anti-Jagged1+anti-Jagged2 antibodies 24hours and 96 hours after the first aerosol challenge.

FIG. 23A shows periodic acid-Schiff staining of lung airways in the micetreated with anti-Jagged1, anti-Jagged2, anti-Jagged1+anti-Jagged2, orcontrol antibody. FIG. 23B shows quantification of the goblet cells inthe airways of the different treatment groups. An abundance of gobletcells are evident in the control and anti-Jagged2 groups. Few gobletcells were present in the anti-Jagged1 group and practically no gobletcells were detected in the anti-Jagged1+anti-Jagged2 group. FIG. 23Cshows the inflammation index as assessed by Haematoxylin Eosin (H&E)staining. Treatment with either Jagged1 or -2 blocking antibody does notaffect inflammation in the lungs.

Jagged1-induced Notch signaling biases cell fate in the airways towardsa secretory cell (including goblet cell) fate and away from a ciliatedcell fate. Jagged1 signaling is important for maintaining the secretorycell fate, and inhibition of Jagged1 signaling prevented goblet cellmetaplasia. We also showed that the club cell-to-ciliated cellconversion is direct and did not involve cell division (data not shown).Club cells give rise to goblet cells in the lung. Thistransdifferentiation of one cell type to another occurred in the adultlung and is distinct from cell fate choices that involve progenitor celldivision, such as after damage or during development. Goblet cellmetaplasia or excess mucus is a hallmark of several airway diseases,such as asthma, cystic fibrosis, COPD and Barrett's esophagus. TheseJagged inhibition results provide the basis for therapeutic applicationsinvolving use of Jagged1 or Jagged2 inhibitors for prevention orreversal of goblet cell metaplasia and for treatment of conditionscharacterized by excess mucus, as in airway diseases (e.g., asthma,COPD, cystic fibrosis) and Barrett's esophagus.

Example 14: Specific Binding of Anti-Jagged1 and Anti-Jagged2 Antibodies

Antibodies A-2 (FIG. 24, left panel) and C-1 (FIG. 24, right panel) weretested for binding to recombinant purified Notch ligands human Jagged1(hJag-1), human Jagged2 (hJag-2), murine Jagged2 (mJag-2), humanDelta-like 1 (hDLL1), murine Delta-like 1 (mDLL1), and human Delta-like4 (hDLL4) using a standard enzyme-linked immunosorbent assay (ELISA). 1μg/ml of Notch ligand protein (as indicated) in PBS, pH7.4, was coatedonto ELISA plates (Nunc Maxisorp) at 40° C. overnight. Plates wereblocked with Casein blocker in PBS (Pierce) for one hour at roomtemperature. Serial 3-fold dilutions of antibody IgGs (as indicated) inPBST buffer (PBT buffer (PBS+0.05% (v/v) Tween 20) with 0.5% (w/v) BSA)were added to the plates and incubated for one hour at room temperature.The plates were then washed with PBST and bound antibodies were detectedwith peroxidase-conjugated goat anti-human Fab specific IgG (Sigma). TMBsubstrate (3,3′,5,5′-tetramethylbenzidine) was used and absorbance at630 nM was read using a standard ELISA plate reader. Absorbance wasplotted against concentrations of IgGs using KaleidaGraph (SynergySoftware). FIG. 24 shows the results, with A₆₃₀ on the y-axisrepresenting the extent of binding.

Antibody A2 bound human and murine Jagged1, but did not bind humanJagged 2, murine Jagged2, human DLL1, murine DLL1, human DLL4, or murineDLL4 (FIG. 24, left panel). Antibody C1 bound human and murine Jagged1,human and murine DLL1, human and murine Jagged2, but did not bind humanor murine DLL4 (FIG. 24, right panel).

Antibody binding affinities and rate constants were measured by SurfacePlasmon Resonance (SPR) using a BIAcore™-T200 instrument. Human IgG1antibodies were captured by mouse anti-human IgG coated on the CMSsensor chip to achieve approximately 150 response units (RU). Forkinetic or affinity measurements, four-fold serial dilutions of humanJagged1, murine Jagged1, human Jagged2, murine Jagged2, human DLL1,murine DLL1, human DLL4, murine DLL4, and rat Jagged1 were injected inHBS-T buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% v/v SurfactantP20, GE Healthcare) at 25° C. with a flow rate of 30 ml/min. The ligandfragments were the DSL-EGF1-4 fragments, except for rat Jagged1, whichwas purchased from R&D Systems. For kinetic analysis, association rates(k_(on)) and dissociation rates (k_(off)) were calculated using a simpleone-to-one Langmuir binding model (BIAcore T200 Evaluation Softwareversion 2.0). The equilibrium dissociation constant (k_(d)) wascalculated as the ratio k_(off)/k_(on). For affinity analysis, K_(d) wascalculated using a Steady State Affinity model (BIAcore T200 EvaluationSoftware version 2.0).

Table 3 summarizes the binding constants for antibodies A-2, B-3, C-1,A-1, and A-1 S101T binding to purified human Jagged1, murine Jagged1,human Jagged2, murine Jagged2, human DLL1, murine DLL1, human DLL4,murine DLL4, and/or rat Jagged1. n.d.=not detected, n.t.=not tested.Antibodies A-2, A-1, and A-1-S101T.NG (antibody A-1S101T with N297Gmutation) specifically bound to human and murine Jagged1 with highaffinity. Antibodies A-1 and A-1-S101T.NG bound rat Jagged1 with highaffinity also. Antibody B-3 specifically bound to human and murineJagged2 with high affinity. Antibody C-1 specifically bound to human andmurine Jagged1 and Jagged 2. Antibodies B-3 and C-1, but not antibodyA-2, showed some binding to human and mouse DLL1. None of the antibodiestested bound human or mouse DLL4.

TABLE 3 Binding constants for anti-Jagged antibodies A-2 B-3 C-1 k_(on)k_(off) K_(d) k_(on) k_(off) K_(d) k_(on) k_(off) K_(d) (1/Ms) (1/s) (M)(1/Ms) (1/s) (M) (1/Ms) (1/s) (M) hJag1 2.42E+05 9.23E−05 3.82E−10 n.d.n.d. >1.00E−06* 2.90E+05 9.00E−05 3.10E−10 mJag1 3.85E+05 5.77E−051.50E−10 n.d. n.d. >1.00E−06* 8.69E+05 1.54E−04 1.77E−10 hJag2 n.d. n.d.n.d. 4.58E+06 1.88E−04 4.10E−11 7.64E+06 5.56E−04 7.28E−11 mJag2 n.d.n.d. n.d. 7.71E+05 4.40E−05 5.71E−11 1.16E+06 8.03E−05 6.92E−11 hDLL1n.d. n.d. n.d. n.d. n.d.  3.49E−07* n.d. n.d.  7.83E−08* mDLL1 n.d. n.d.n.d. n.d. n.d.  7.23E−08* n.d. n.d.  5.55E−08* hDLL4 n.d. n.d. n.d. n.d.n.d. n.d. n.d. n.d. n.d. mDLL4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.n.d. rJag1 n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. A-1 A-1.S101T.NGk_(on) k_(off) K_(d) k_(on) k_(off) K_(d) (1/Ms) (1/s) (M) (1/Ms) (1/s)(M) hJag1 2.91E+04 2.25E−04 7.75E−09 2.09E+04 5.88E−04 2.82E−08 mJag11.59E+05 7.42E−05 4.57E−01 9.51E+04 6.22E−04 6.54E−09 hJag2 n.d. n.d.n.d. n.d. n.d. n.d. mJag2 n.d. n.d. n.d. n.d. n.d. n.d. hDLL1 n.t. n.t.n.t. n.t. n.t. n.t. mDLL1 n.t. n.t. n.t. n.t. n.t. n.t. hDLL4 n.t. n.t.n.t. n.t. n.t. n.t. mDLL4 n.t. n.t. n.t. n.t. n.t. n.t. rJag1 4.34E+049.80E−07 2.26E−11 4.55E+04 1.97E−04 4.32E−09

Example 15: Pharmacokinetics of Anti-Jagged1 A-1-S101T Antibody

The pharmacokinetic profile of anti-Jagged1 A-1-S101T antibody followinga single intravenous injection at 1, 10, and 100 mg/kg were evaluated infemale Balb/c nude mice (Charles River Laboratories, Hollister, Calif.).The mice were 5-8 weeks old and weighed approximately 17.3-21.8 g. Serumsamples were collected and antibody concentrations were analyzed byspecific enzyme linked immunosorbent assays (ELISA). The specific ELISAwas coated with JAG1 extracellular domain-Histidine and detected withgoat anti-human Fc. The assay sensitivity has a less than standard valueof 6.25 ng/mL. Pharmacokinetic parameters were estimated using anon-compartmental model with Phoenix™ WinNonlin® (v.6.3; PharsightCorporation; Mountain View, Calif.). All PK analysis was based on naïvepool of individual animal data.

A greater than dose proportional increase in exposure was observedfollowing IV administration of anti-Jagged1 A-1-S101T antibody withinthe dose range of 1 and 100 mg/kg, suggesting a target-mediatedclearance mechanism of the antibody (FIG. 25 and Table 4). The clearancevalues ranged from approximately 13 to 75 mL/day/kg.

TABLE 4 Pharmacokinetic properties of anti-Jagged1 A-1-S101T antibodyC_(max) AUC_(last) AUC_(inf) CL V_(ss) Treatment ELISA (μg/mL)(day*μg/mL) (day*μg/mL) (mL/day/kg) (mL/kg) 1 mg/kg specific  14.5 ±0.497  13.3 ± 0.552 13.3 75.0 80.7 anti-JAG1 10 mg/kg specific 160 ±2.00 458 ± 19.3 458 21.8 87.6 anti-JAG1 100 mg/kg specific 1480 ± 68.4 7220 ± 443  7410 13.5 115 anti-JAG1

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Table of Sequences SEQ ID NO Description Sequence  1 Human Jag 1MRSPRTRGRS GRPLSLLLAL LCALRAKVCG ASGQFELEIL SMQNVNGELQNGNCCGGARN PGDRKCTRDE CDTYFKVCLK EYQSRVTAGG PCSFGSGSTPVIGGNTFNLK ASRGNDRNRI VLPFSFAWPR SYTLLVEAWD SSNDTVQPDSIIEKASHSGM INPSRQWQTL KQNTGVAHFE YQIRVTCDDY YYGFGCNKFCRPRDDFFGHY ACDQNGNKTC MEGWMGPECN RAICRQGCSP KHGSCKLPGDCRCQYGWQGL YCDKCIPHPG CVHGICNEPW QCLCETNWGG QLCDKDLNYCGTHQPCLNGG TCSNTGPDKY QCSCPEGYSG PNCEIAEHAC LSDPCHNRGSCKETSLGFEC ECSPGWTGPT CSTNIDDCSP NNCSHGGTCQ DLVNGFKCVCPPQWTGKTCQ LDANECEAKP CVNAKSCKNL IASYYCDCLP GWMGQNCDININDCLGQCQN DASCRDLVNG YRCICPPGYA GDHCERDIDE CASNPCLNGGHCQNEINRFQ CLCPTGFSGN LCQLDIDYCE PNPCQNGAQC YNRASDYFCKCPEDYEGKNC SHLKDHCRTT PCEVIDSCTV AMASNDTPEG VRYISSNVCGPHGKCKSQSG GKFTCDCNKG FTGTYCHENI NDCESNPCRN GGTCIDGVNSYKCICSDGWE GAYCETNIND CSQNPCHNGG TCRDLVNDFY CDCKNGWKGKTCHSRDSQCD EATCNNGGTC YDEGDAFKCM CPGGWEGTTC NIARNSSCLPNPCHNGGTCV VNGESFTCVC KEGWEGPICA QNTNDCSPHP CYNSGTCVDGDNWYRCECAP GFAGPDCRIN INECQSSPCA FGATCVDEIN GYRCVCPPGHSGAKCQEVSG RPCITMGSVI PDGAKWDDDC NTCQCLNGRI ACSKVWCGPRPCLLHKGHSE CPSGQSCIPI LDDQCFVHPC TGVGECRSSS LQPVKTKCTSDSYYQDNCAN ITFTFNKEMM SPGLTTEHIC SELRNLNILK NVSAEYSIYIACEPSPSANN EIHVAISAED IRDDGNPIKE ITDKIIDLVS KRDGNSSLIAAVAEVRVQRR PLKNRTDFLV PLLSSVLTVA WICCLVTAFY WCLRKRRKPGSHTHSASEDN TTNNVREQLN QIKNPIEKHG ANTVPIKDYE NKNSKMSKIRTHNSEVEEDD MDKHQQKARF AKQPAYTLVD REEKPPNGTP TKHPNWTNKQDNRDLESAQS LNRMEYIV  2 Murine Jag 1MRSPRTRGRP GRPLSLLLAL LCALRAKVCG ASGQFELEIL SMQNVNGELQNGNCCGGVRN PGDRKCTRDE CDTYFKVCLK EYQSRVTAGG PCSFGSGSTPVIGGNTFNLK ASRGNDRNRI VLPFSFAWPR SYTLLVEAWD SSNDTIQPDSIIEKASHSGM INPSRQWQTL KQNTGIAHFE YQIRVTCDDH YYGFGCNKFCRPRDDFFGHY ACDQNGNKTC MEGWMGPDCN KAICRQGCSP KHGSCKLPGDCRCQYGWQGL YCDKCIPHPG CVHGTCNEPW QCLCETNWGG QLCDKDLNYCGTHQPCLNRG TCSNTGPDKY QCSCPEGYSG PNCEIAEHAC LSDPCHNRGSCKETSSGFEC ECSPGWTGPT CSTNIDDCSP NNCSHGGTCQ DLVNGFKCVCPPQWTGKTCQ LDANECEAKP CVNARSCKNL IASYYCDCLP GWMGQNCDININDCLGQCQN DASCRDLVNG YRCICPPGYA GDHCERDIDE CASNPCLNGGHCQNEINRFQ CLCPTGFSGN LCQLDIDYCE PNPCQNGAQC YNRASDYFCKCPEDYEGKNC SHLKDHCRTT TCEVIDSCTV AMASNDTPEG VRYISSNVCGPHGKCKSQSG GKFTCDCNKG FTGTYCHENI NDCESNPCKN GGTCIDGVNSYKCICSDGWE GAHCENNIND CSQNPCHYGG TCRDLVNDFY CDCKNGWKGKTCHSRDSQCD EATCNNGGTC YDEVDTFKCM CPGGWEGTTC NIARNSSCLPNPCHNGGTCV VNGDSFTCVC KEGWEGPICT QNTNDCSPHP CYNSGTCVDGDNWYRCECAP GFAGPDCRIN INECQSSPCA FGATCVDEIN GYQCICPPGHSGAKCHEVSG RSCITMGRVI LDGAKWDDDC NTCQCLNGRV ACSKVWCGPRPCRLHKSHNE CPSGQSCIPV LDDQCFVRPC TGVGECRSSS LQPVKTKCTSDSYYQDNCAN ITFTFNKEMM SPGLTTEHIC SELRNLNILK NVSAEYSIYIACEPSLSANN EIHVAISAED IRDDGNPVKE ITDKIIDLVS KRDGNSSLIAAVAEVRVQRR PLKNRTDFLV PLLSSVLTVA WVCCLVTAFY WCVRKRRKPSSHTHSAPEDN TTNNVREQLN QIKNPIEKHG ANTVPIKDYE NKNSKMSKIRTHNSEVEEDD MDKHQQKVRF AKQPVYTLVD REEKAPSGTP TKHPNWTNKQDNRDLESAQS LNRMEYIV  3 Human Jag2MRAQGRGRLP RRLLLLLALW VQAARPMGYF ELQLSALRNV NGELLSGACCDGDGRTTRAG GCGHDECDTY VRVCLKEYQA KVTPTGPCSY GHGATPVLGGNSFYLPPAGA AGDRARARAR AGGDQDPGLV VIPFQFAWPR SFTLIVEAWDWDNDTTPNEE LLIERVSHAG MINPEDRWKS LHFSGHVAHL ELQIRVRCDENYYSATCNKF CRPRNDFFGH YTCDQYGNKA CMDGWMGKEC KEAVCKQGCNLLHGGCTVPG ECRCSYGWQG RFCDECVPYP GCVHGSCVEP WQCNCETNWGGLLCDKDLNY CGSHHPCTNG GTCINAEPDQ YRCTCPDGYS GRNCEKAEHACTSNPCANGG SCHEVPSGFE CHCPSGWSGP TCALDIDECA SNPCAAGGTCVDQVDGFECI CPEQWVGATC QLDANECEGK PCLNAFSCKN LIGGYYCDCIPGWKGINCHI NVNDCRGQCQ HGGTCKDLVN GYQCVCPRGF GGRHCELERDECASSPCHSG GLCEDLADGF HCHCPQGFSG PLCEVDVDLC EPSPCRNGARCYNLEGDYYC ACPDDFGGKN CSVPREPCPG GACRVIDGCG SDAGPGMPGTAASGVCGPHG RCVSQPGGNF SCICDSGFTG TYCHENIDDC LGQPCRNGGTCIDEVDAFRC FCPSGWEGEL CDTNPNDCLP DPCHSRGRCY DLVNDFYCACDDGWKGKTCH SREFQCDAYT CSNGGTCYDS GDTFRCACPP GWKGSTCAVAKNSSCLPNPC VNGGTCVGSG ASFSCICRDG WEGRTCTHNT NDCNPLPCYNGGICVDGVNW FRCECAPGFA GPDCRINIDE CQSSPCAYGA TCVDEINGYRCSCPPGRAGP RCQEVIGFGR SCWSRGTPFP HGSSWVEDCN SCRCLDGRRDCSKVWCGWKP CLLAGQPEAL SAQCPLGQRC LEKAPGQCLR PPCEAWGECGAEEPPSTPCL PRSGHLDNNC ARLTLHFNRD HVPQGTTVGA ICSGIRSLPATRAVARDRLL VLLCDRASSG ASAVEVAVSF SPARDLPDSS LIQGAAHAIVAAITQRGNSS LLLAVTEVKV ETVVTGGSST GLLVPVLCGA FSVLWLACVVLCVWWTRKRR KERERSRLPR EESANNQWAP LNPIRNPIER PGGHKDVLYQCKNFTPPPRR ADEALPGPAG HAAVREDEED EDLGRGEEDS LEAEKFLSHKFTKDPGRSPG RPAHWASGPK VDNRAVRSIN EARYAGKE  4 Murine Jag2MRARGWGRLP RRLLLLLVLC VQATRPMGYF ELQLSALRNV NGELLSGACCDGDGRTTRAG GCGRDECDTY VRVCLKEYQA KVTPTGPCSY GYGATPVLGGNSFYLPPAGA AGDRARARSR TGGHQDPGLV VIPFQFAWPR SFTLIVEAWDWDNDTTPDEE LLIERVSHAG MINPEDRWKS LHFSGHVAHL ELQIRVRCDENYYSATCNKF CRPRNDFFGH YTCDQYGNKA CMDGWMGKEC KEAVCKQGCNLLHGGCTVPG ECRCSYGWQG KFCDECVPYP GCVHGSCVEP WHCDCETNWGGLLCDKDLNY CGSHHPCVNG GTCINAEPDQ YLCACPDGYL GKNCERAEHACASNPCANGG SCHEVPSGFE CHCPSGWSGP TCALDIDECA SNPCAAGGTCVDQVDGFECI CPEQWVGATC QLDANECEGK PCLNAFSCKN LIGGYYCDCLPGWKGINCQI NINDCHGQCQ HGGTCKDLVN GYQCVCPRGF GGRHCELEYDKCASSPCRRG GICEDLVDGF RCHCPRGLSG LHCEVDMDLC EPSPCLNGARCYNLEGDYYC ACPEDFGGKN CSVPRDTCPG GACRVIDGCG FEAGSRARGVAPSGICGPHG HCVSLPGGNF SCICDSGFTG TYCHENIDDC MGQPCRNGGTCIDEVDSFRC FCPSGWEGEL CDINPNDCLP DPCHSRGRCY DLVNDFYCACDDGWKGKTCH SREFQCDAYT CSNGGTCYDS GDTFRCACPP GWKGSTCTIAKNSSCVPNPC VNGGTCVGSG DSFSCICRDG WEGRTCTHNT NDCNPLPCYNGGICVDGVNW FRCECAPGFA GPDCRINIDE CQSSPCAYGA TCVDEINGYRCSCPPGRSGP RCQEVVIFTR PCWSRGMSFP HGSSWMEDCN SCRCLDGHRDCSKVWCGWKP CLLSGQPSDP SAQCPPGQQC QEKAVGQCLQ PPCENWGECTAEEPLPPSTP CQPRSSHLDN NCARLTLRFN RDQVPQGTTV GAICSGIRALPATRAAAHDR LLLLLCDRAS SGASAVEVAM SFSPARDLPD SSLIQSTAHAIVAAITQRGN SSLLLAVTEV KVETVVMGGS STGLLVPVLC SVFSVLWLACVVICVWWTRK RRKERERSRL PRDESTNNQW APLNPIRNPI ERPGGSGLGTGGHKDILYQC KNFTPPPRRA GEALPGPAGH GAGGEDEEDE ELSRGDGDSPEAEKFISHKF TKDPSCSLGR PACWAPGPKV DNRAVRSTKD VRRAGRE  5 Murine Jag1-DSL-ADLGSQFELE ILSMQNVNGE LQNGNCCGGV RNPGDRKCTR DECDTYFKVC EGF1-4 (mouseLKEYQSRVTA GGPCSFGSGS TPVIGGNTFN LKASRGNDRN RIVLPFSFAW Jag1 antigen)PRSYTLLVEA WDSSNDTIQP DSIIEKASHS GMINPSRQWQ TLKQNTGIAHFEYQIRVTCD DHYYGFGCNK FCRPRDDFFG HYACDQNGNK TCMEGWMGPDCNKAICRQGC SPKHGSCKLP GDCRCQYGWQ GLYCDKCIPH PGCVHGTCNEPWQCLCETNW GGQLCDKDLN YCGTHQPCLN RGTCSNTGPD KYQCSCPEGYSGPNCEIAEH ACLSDPCHNR GSCKETSSGF ECECSPGWTG PTCSTNIDDE FGLVPRGSGH HHHHH 6 human Jag1-DSL-QFELEILSMQ NVNGELQNGN CCGGARNPGD RKCTRDECDT YFKVCLKEYQ EGF1-4 (humanSRVTAGGPCS FGSGSTPVIG GNTFNLKASR GNDRNRIVLP FSFAWPRSYT JAG1 antigen)LLVEAWDSSN DTVQPDSIIE KASHSGMINP SRQWQTLKQN TGVAHFEYQIRVTCDDYYYG FGCNKFCRPR DDFFGHYACD QNGNKTCMEG WMGPECNRAICRQGCSPKHG SCKLGDCRCQ YGWQGLYCDK CIPHPGCVHG ICNEPWQCLCETNWGGQLCD KDLNYCGTHQ PCLNGGTCSN TGPDKYQCSC PEGYSGPNCEIAEHACLSDP CHNRGSCKET SLGFECECSP GWTGPTCSTN IDD  7 murine Jag2-DSL-ADLGSMGYFE LQLSALRNVN GELLSGACCD GDGRTTRAGG CGRDECDTYV EGF1-4 (mouseRVCLKEYQAK VTPTGPCSYG YGATPVLGGN SFYLPPAGAA GDRARARSRT Jag2 antigen)GGHQDPGLVV IPFQFAWPRS FTLIVEAWDW DNDTTPDEEL LIERVSHAGMINPEDRWKSL HFSGHVAHLE LQIRVRCDEN YYSATCNKFC RPRNDFFGHYTCDQYGNKAC MDGWMGKECK EAVCKQGCNL LHGGCTVPGE CRCSYGWQGKFCDECVPYPG CVHGSCVEPW HCDCETNWGG LLCDKDLNYC GSHHPCVNGGTCINAEPDQY LCACPDGYLG KNCERAEHAC ASNPCANGGS CHEVPSGFECHCPSGWNGPT CALDIDEEFG LVPRGSGHHH HHH  8 human Jag2-DSL-ARPMGYFELQ LSALRNVNGE LLSGACCDGD GRTTRAGGCG HDECDTYVRV EGF1-4 (humanCLKEYQAKVT PTGPCSYGHG ATPVLGGNSF YLPPAGAAGD RARARARAGG Jag2 antigen)DQDPGLWIP FQFAWPRSFT LIVEAWDWDN DTTPNEELLI ERVSHAGMINPEDRWKSLHF SGHVAHLELQ IRVRCDENYY SATCNKFCRP RNDFFGHYTCDQYGNKACMD GWMGKECKEA VCKQGCNLLH GGCTVPGECR CSYGWQGRFCDECVPYPGCV HGSCVEPWQC NCETNWGGLL CDKDLNYCGS HHPCTNGGTCINAEPDQYRC TCPDGYSGRN CEKAEHACTS NPCANGGSCH EVPSGFECHCPSGWSGPTCA LDIDEEFGLV PRGSGHHHHH H  9 Antibody A heavyEVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW chain variableITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG regionSWFAYWGQGT LVTVSS 10 Antibody A lightDIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS chain variableASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYTTPPTFGQ region GTKVEIK 11Antibody A heavy GFTFSNYGIH chain hypervariable region 1 (HVR- H1) 12Antibody A HVR- WITPDGGYTDYADSVKG H2 13 Antibody A HVR- AGSWFAY H3 14Antibody A light RASQDVSTAVA chain hypervariable region 1 (HVR- L1) 15Antibody A HVR- SASFLYS L2 16 Antibody A HVR- QQSYTTPPT L3 17Antibody A-1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGWheavy chain ITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAGvariable region SLFAYWGQGT LVTVSS 18 Antibody A-1DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS light chainASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYTTATTFGQ variable regionGTKVEIK 19 Antibody A-1 GFTFSNYGIH HVR-H1 20 Antibody A-1WITPDGGYTDYADSVKG HVR-H2 21 Antibody A-1 AGSLFAY HVR-H3 22 Antibody A-1RASQDVSTAVA HVR-L1 23 Antibody A-1 SASFLYS HVR-L2 24 Antibody A-1QQYYTTATT HVR-L3 25 Antibody A-2EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW heavy chainITGNGGYSDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG variable regionSWFAYWGQGT LVTVSS 26 Antibody A-2DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS light chainASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYTTPPTFGQ variable regionGTKVEIK 27 Antibody A-2 GFTFSNYGIH HVR-H1 28 Antibody A-2WITGNGGYSDYADSVKG HVR-H2 29 Antibody A-2 AGSWFAY HVR-H3 30 Antibody A-2RASQDVSTAVA HVR-L1 31 Antibody A-2 SASFLYS HVR-L2 32 Antibody A-2QQSYTTPPT HVR-L3 33 Antibody A-1EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW (S101T) heavyITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG chain variableTLFAYWGQGT LVTVSS region 34 Antibody A-1DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS (S101T) lightASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYTTATTFGQ chain variableGTKVEIK region 35 Antibody A-1 GFTFSNYGIH (S101T) HVR- H1 36Antibody A-1 WITPDGGYTDYADSVKG (S101T) HVR- H2 37 Antibody A-1 AGTLFAY(S101T) HVR- H3 38 Antibody A-1 RASQDVSTAVA (S101T) HVR-L1 39Antibody A-1 SASFLYS (S101T) HVR-L2 40 Antibody A-1 QQYYTTATT(S101T) HVR-L3 41 Antibody B-3EVQLVESGGG LVQPGGSLRL SCAASGFTFT SYDIHWVRQA PGKGLEWVGG heavy chainISPADGDTDY ANSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARND variable regionYDVRFVGSGM DYWGQGTLVT VSS 42 Antibody B-3DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS light chainASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SFTAPPTFGQ variable regionGTKVEIK 43 Antibody A, A-1, DIQMTQSPSS LSASVGDRVT ITC A-2, A-1(S101T),B-3 light chain framework 1 (LC- FR1) 44 Antibody A, A-1,WYQQKP GKAPKLLIY A-2, A-1(S101T), B-3 LC-FR2 45 Antibody A, A-1,GVPS RFSGSGSGTD FTLTISSLQP EDFATYYC A-2, A-1(S101T), B-3 LC-FR3 46Antibody A, A-1, FGQ GTKVEIK A-2, A-1(S101T), B-3 LC-FR4 47Antibody A, A-1, EVQLVESGGG LVQPGGSLRL SCAAS A-2, A-1(S101T),B-3 heavy chain framework 1(HC- FR1) 48 Antibody A,A-1, WVRQA PGKGLEWVGA-2, A-1(S101T), B-3 HC-FR2 49 Antibody A, A-1,RFTI SADTSKNTAY LQMNSLRAED TAVYYCAR A-2, A-1(S101T), B-3 HC-FR3 50Antibody A, A-1, WGQGT LVTVSS A-2, A-1(S101T), B-3 HC-FRA 51Antibody A-1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW(S101T) IgG1 ITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAGheavy chain TLFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFPEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 52 Antibody A-1EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW (S101T) IgG1ITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG heavy chainTLFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP N297GEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYGSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 53 Antibody A-1DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS (S101T) lightASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYTTATTFGQ chain; antibody GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV A-1 light chainDNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC54 Antibody A-1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW(S101X) heavy ITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAGchain variable XLFAYWGQGT LVTVSS regionX is any amino acid other than S. 55 Antibody A-1 AGXLFAY (S101X) HVR-H3X is any amino acid other than S. 56 Antibody A-1EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW (S101X)IgG1ITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG heavy chainXLFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFPEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGKX is any amino acid other than S. 57 Antibody A-1EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW (S101X)IgG1ITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG heavy chainXLFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP N297GEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYGSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGKX is any amino acid other than S. 58 AntibodyEVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW A(S101X) heavyITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG chain variableXWFAYWGQGT LVTVSS region X is any amino acid other than S. 59 AntibodyAGXWFAY A(S101X) HVR- X is any amino acid other than S. H3; Antibody A-2(S101X) HVR- H3 62 Antibody A-2EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW (S101X) heavyITGNGGYSDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG chain variableXWFAYWGQGT LVTVSS region X is any amino acid other than S. 63Antibody A-1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGWIgG1 heavy chain ITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAGN297G SLFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFPEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYGSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 64 Antibody AGTWFAYA(S101T) HVR- H3; Antibody A- 2(S101T) HVR- H3 65 AntibodyEVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW A(S101T) heavyITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG chain variableTWFAYWGQGT LVTVSS region 66 Antibody A-EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW 2(S101T) heavyITGNGGYSDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG chain variableTWFAYWGQGT LVTVSS region 67 AntibodyEVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW A(S101X) IgG1ITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG heavy chainXWFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP N297GEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYGSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGKX is any amino acid other than S. 68 Antibody A-2EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW (S101X) IgG1ITGNGGYSDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG heavy chainXWFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP N297GEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYGSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGKX is any amino acid other than S. 69 AntibodyEVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW A(S101T) IgG1ITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG heavy chainTWFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP N297GEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYGSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 70 Antibody A-EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW 2(S101T) IgG1ITGNGGYSDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG heavychainTWFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP N297GEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYGSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 71 HVR-H2WIT(X1)(X2)GGY(X3)DYADSVKG consensusX1 is P or G; X2 is D or N; X3 is T or S 77 HVR-H3 AG(X1)(X2)FAYconsensus X1 is S or T; X2 is W or L 72 HVR-H3 AG(X1)(X2)FAYconsensus (101X) X1 is any amino acid other than S; X2 is W or L 73HVR-H3 AGT(X1)FAY consensus (101T) X1 is W or L 74 HVR-L3QQ(X1)YTT(X2)(X3)T consensus X1 is S or Y; X2 is P or A; X3 is P or T 75Antibody A light DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYSchain ASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYTTPPTFGQGTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC76 Antibody A-2 DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYSlight chain ASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYTTPPTFGQGTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC78 Antibody A, A-1, NYGIH A-1(S101T), A-2 alternative HVR- H1 79Antibody EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGWA(S101T) IgG1 ITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAGheavy chain TWFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFPEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 80 Antibody A-EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW 2(S101T) IgG1ITGNGGYSDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAG heavy chainTWFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFPEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK 81 Ainibody A-1EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYGIHWVRQA PGKGLEWVGW IgG1 heavy chainITPDGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARAGSLFAYWGQGT LVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFPEPVTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICNVNHKPSNTKV DKKVEPKSCD KTHTCPPCPA PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYRVVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTLPPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSDGSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK

What is claimed is:
 1. A method of treating an individual with cancerassociated with aberrant Notch signaling comprising administering to theindividual an effective amount of an antibody that binds to humanJagged1, wherein the antibody comprises HVR-H1 comprising the amino acidsequence of SEQ ID NO: 35 or 78; HVR-H2 comprising the amino acidsequence of SEQ ID NO: 28 or 36; HVR-H3 comprising the amino acid of SEQID NO: 55 or 59, wherein X is any amino acid other than S; HVR-L1comprising the amino acid sequence of SEQ ID NO: 38; HVR-L2 comprisingthe amino acid sequence of SEQ ID NO: 39; and HVR-L3 comprising theamino acid sequence of SEQ ID NO: 16 or
 40. 2. The method of claim 1,wherein the antibody comprises: a) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 78; HVR-H2 comprising the amino acid sequence ofSEQ ID NO: 36; and HVR-H3 comprising the amino acid sequence of SEQ IDNO: 55; or b) HVR-H1 comprising the amino acid sequence of SEQ ID NO:78; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36; andHVR-H3 comprising the amino acid sequence of SEQ ID NO: 59; or c) HVR-H1comprising the amino acid sequence of SEQ ID NO: 78; HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 28; and HVR-H3 comprising theamino acid sequence of SEQ ID NO:
 59. 3. The method of claim 2, whereinthe antibody comprises: a) HVR-L1 comprising the amino acid sequence ofSEQ ID NO: 38; HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 40; orb) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 38; HVR-L2comprising the amino acid sequence of SEQ ID NO: 39; and HVR-L3comprising the amino acid sequence of SEQ ID NO:
 16. 4. The method ofclaim 1, wherein the antibody comprises: a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 78; HVR-H2 comprising the amino acidsequence of SEQ ID NO: 36; HVR-H3 comprising the amino acid sequence ofSEQ ID NO: 55; HVR-L1 comprising the amino acid sequence of SEQ ID NO:38; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; andHVR-L3 comprising the amino acid sequence of SEQ ID NO: 40; or b) HVR-H1comprising the amino acid sequence of SEQ ID NO: 78; HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 36; HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 59; HVR-L1 comprising the amino acidsequence of SEQ ID NO: 38; HVR-L2 comprising the amino acid sequence ofSEQ ID NO: 39; and HVR-L3 comprising the amino acid sequence of SEQ IDNO: 16; or c) HVR-H1 comprising the amino acid sequence of SEQ ID NO:78; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28; HVR-H3comprising the amino acid sequence of SEQ ID NO: 59; HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 38; HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 39; and HVR-L3 comprising the amino acidsequence of SEQ ID NO:
 16. 5. The method of claim 1, wherein theantibody comprises a VH sequence having at least 95% identity to theamino acid sequence of SEQ ID NO: 54, 58, or
 62. 6. The method of claim1, wherein the antibody comprises a VL sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 10, 26, or
 34. 7. Themethod of claim 1, wherein the antibody comprises: a) a VH sequencehaving at least 95% identity to the amino acid sequence of SEQ ID NO: 54and a VL sequence having at least 95% identity to the amino acidsequence of SEQ ID NO: 34; or b) a VH sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 58 and a VL sequencehaving at least 95% identity to the amino acid sequence of SEQ ID NO:10; or c) a VH sequence having at least 95% identity to the amino acidsequence of SEQ ID NO: 62 and a VL sequence having at least 95% identityto the amino acid sequence of SEQ ID NO:
 26. 8. A method of treating anindividual with cancer associated with aberrant Notch signalingcomprising administering to the individual an effective amount of anantibody that binds human Jagged1, wherein the antibody comprises: a) aVH sequence of SEQ ID NO: 54, wherein X is any amino acid other than S,and a VL sequence of SEQ ID NO: 34; or b) a VH sequence of SEQ ID NO:58, wherein X is any amino acid other than S, and a VL sequence of SEQID NO: 10; or c) a VH sequence of SEQ ID NO: 62, wherein X is any aminoacid other than S, and a VL sequence of SEQ ID NO:
 26. 9. The method ofclaim 8, wherein the antibody comprises a VH sequence of SEQ ID NO: 54and a VL sequence of SEQ ID NO:
 34. 10. The method of claim 9, whereinthe heavy chain comprises the amino acid sequence of SEQ ID NO: 56 andthe light chain comprises the amino acid sequence of SEQ ID NO:
 53. 11.The method of claim 9, wherein the heavy chain comprises the amino acidsequence of SEQ ID NO: 57 and the light chain comprises the amino acidsequence of SEQ ID NO:
 53. 12. The method of claim 1, wherein X is anyamino acid other than S or H.
 13. The method of claim 1, wherein X isselected from A, D, E, G, I, K, L, N, Q, R T, and V.
 14. The method ofclaim 1, wherein X is T.
 15. A method of treating an individual withcancer associated with aberrant Notch signaling comprising administeringto the individual an effective amount of an antibody that binds humanJagged1, wherein the antibody comprises: a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 78; HVR-H2 comprising the amino acidsequence of SEQ ID NO: 36; HVR-H3 comprising the amino acid sequence ofSEQ ID NO: 37; HVR-L1 comprising the amino acid sequence of SEQ ID NO:38; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 39; andHVR-L3 comprising the amino acid sequence of SEQ ID NO: 40; or b) HVR-H1comprising the amino acid sequence of SEQ ID NO: 78; HVR-H2 comprisingthe amino acid sequence of SEQ ID NO: 36; HVR-H3 comprising the aminoacid sequence of SEQ ID NO: 64; HVR-L1 comprising the amino acidsequence of SEQ ID NO: 38; HVR-L2 comprising the amino acid sequence ofSEQ ID NO: 39; and HVR-L3 comprising the amino acid sequence of SEQ IDNO: 16; or c) HVR-H1 comprising the amino acid sequence of SEQ ID NO:78; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 28; HVR-H3comprising the amino acid sequence of SEQ ID NO: 64; HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 38; HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 39; and HVR-L3 comprising the amino acidsequence of SEQ ID NO:
 16. 16. The method of claim 15, wherein theantibody comprises: a) a VH sequence of SEQ ID NO: 33 and a VL sequenceof SEQ ID NO: 34; or b) a VH sequence of SEQ ID NO: 65 and a VL sequenceof SEQ ID NO: 10; or c) a VH sequence of SEQ ID NO: 66 and a VL sequenceof SEQ ID NO:
 26. 17. The method of claim 16, wherein the antibodycomprises a VH sequence of SEQ ID NO: 33 and a VL sequence of SEQ ID NO:34.
 18. The method of claim 1, which is a full length IgG1 antibody. 19.The method of claim 18, wherein the antibody substantially lackseffector function.
 20. The method of claim 18, wherein the heavy chaincomprises a N297G or N297A mutation.
 21. A method of treating anindividual with cancer associated with aberrant Notch signalingcomprising administering to the individual an effective amount of anantibody that binds human Jagged1, wherein the heavy chain comprises theamino acid sequence of SEQ ID NO: 51 and the light chain comprises theamino acid sequence of SEQ ID NO:
 53. 22. A method of treating anindividual with cancer associated with aberrant Notch signalingcomprising administering to the individual an effective amount of anantibody that binds human Jagged1, wherein the heavy chain comprises theamino acid sequence of SEQ ID NO: 52 and the light chain comprises theamino acid sequence of SEQ ID NO:
 53. 23. The method of claim 16,wherein the antibody comprises: a) a heavy chain comprising the aminoacid sequence of SEQ ID NO: 69 and a light chain comprising the aminoacid sequence of SEQ ID NO: 75; or b) a heavy chain comprising the aminoacid sequence of SEQ ID NO: 70 and a light chain comprising the aminoacid sequence of SEQ ID NO: 76; or c) a heavy chain comprising the aminoacid sequence of SEQ ID NO: 79 and a light chain comprising the aminoacid sequence of SEQ ID NO: 75; or d) a heavy chain comprising the aminoacid sequence of SEQ ID NO: 80 and a light chain comprising the aminoacid sequence of SEQ ID NO:
 76. 24. The method of claim 1, wherein theantibody is an antagonist of Jagged1-mediated signaling.
 25. The methodof claim 1, wherein the antibody does not bind human Jagged2.
 26. Themethod of claim 1, wherein the antibody does not bind human DLL4. 27.The method of claim 1, wherein the antibody does not bind human DLL1.28. The method of claim 1, wherein the antibody does not bind murineJagged2, murine DLL4, and/or murine DLL1.
 29. The method of claim 1,wherein the antibody reduces tumor growth in a mouse xenograft modelwithout causing weight loss.
 30. The method of claim 29, wherein themouse xenograft model is a liver cancer xenograft model.
 31. The methodof claim 29, wherein tumor growth is reduced by at least 50 AUC/day TGI%.
 32. The method of claim 1, wherein the cancer is selected from breastcancer, lung cancer, brain cancer, cervical cancer, colon cancer, livercancer, bile duct cancer, pancreatic cancer, skin cancer, B-cellmalignancies, and T-cell malignancies.
 33. The method of claim 8,wherein the cancer is selected from breast cancer, lung cancer, braincancer, cervical cancer, colon cancer, liver cancer, bile duct cancer,pancreatic cancer, skin cancer, B-cell malignancies, and T-cellmalignancies.
 34. The method of claim 15, wherein the cancer is selectedfrom breast cancer, lung cancer, brain cancer, cervical cancer, coloncancer, liver cancer, bile duct cancer, pancreatic cancer, skin cancer,B-cell malignancies, and T-cell malignancies.
 35. The method of claim16, wherein the cancer is selected from breast cancer, lung cancer,brain cancer, cervical cancer, colon cancer, liver cancer, bile ductcancer, pancreatic cancer, skin cancer, B-cell malignancies, and T-cellmalignancies.
 36. The method of claim 21, wherein the cancer is selectedfrom breast cancer, lung cancer, brain cancer, cervical cancer, coloncancer, liver cancer, bile duct cancer, pancreatic cancer, skin cancer,B-cell malignancies, and T-cell malignancies.
 37. The method of claim22, wherein the cancer is selected from breast cancer, lung cancer,brain cancer, cervical cancer, colon cancer, liver cancer, bile ductcancer, pancreatic cancer, skin cancer, B-cell malignancies, and T-cellmalignancies.